From 5c44f761284c0ad77bcf73f8040c779cc4f5ce6c Mon Sep 17 00:00:00 2001
From: Brad Langhorst <langhorst@neb.com>
Date: Mon, 7 Sep 2026 22:06:29 -0400
Subject: [PATCH 1/4] switches to bamslice, fgumi zipper, and picodup, with
 performance improvements across the board (2.4x faster)

---
 .github/workflows/test.yml           |  36 +++-
 BENCHMARK_RESULTS.md                 |  40 ++++
 README.md                            |   8 +-
 conf/base.config                     |  14 ++
 conf/references.config               |  12 +-
 conf/test.config                     |   4 +
 main.nf                              |  49 ++---
 modules/align_reads.nf               |  32 ---
 modules/convert_methylkit_to_bed.nf  |  48 ++---
 modules/fastp.nf                     |  38 ----
 modules/fastqc.nf                    |  18 +-
 modules/gc_bias.nf                   |  11 +-
 modules/idx_stats.nf                 |   2 +-
 modules/insert_size_metrics.nf       |   2 +-
 modules/merge_and_mark_duplicates.nf |  50 -----
 modules/merge_and_picodup.nf         |  42 ++++
 modules/methyldackel_mbias.nf        |   3 +-
 modules/picard_metrics.nf            |   2 +-
 modules/tasmanian.nf                 |  15 +-
 modules/trim_and_align.nf            |  59 +++++
 nextflow.config                      |  20 +-
 tests/main.nf.test                   |  42 ++--
 tests/main.nf.test.snap              | 312 +++++++++++++--------------
 23 files changed, 465 insertions(+), 394 deletions(-)
 create mode 100644 BENCHMARK_RESULTS.md
 delete mode 100644 modules/align_reads.nf
 delete mode 100644 modules/fastp.nf
 delete mode 100644 modules/merge_and_mark_duplicates.nf
 create mode 100644 modules/merge_and_picodup.nf
 create mode 100644 modules/trim_and_align.nf

diff --git a/.github/workflows/test.yml b/.github/workflows/test.yml
index 95628e8..c8ba2c0 100644
--- a/.github/workflows/test.yml
+++ b/.github/workflows/test.yml
@@ -8,7 +8,8 @@ on:
     branches:
         - master
   schedule:
-    - cron: '0 0 1 * *'
+    - cron: '0 6 * * *'
+  workflow_dispatch:
 
 jobs:
   test:
@@ -25,7 +26,38 @@ jobs:
           micromamba-version: '1.5.10-0' # any version from https://github.com/mamba-org/micromamba-releases
           post-cleanup: 'all'
 
-      - name: Setup Nextflow latest-edge
+      - name: Setup Nextflow (minimum supported version)
+        uses: nf-core/setup-nextflow@v2
+        with:
+          version: '25.04.0'
+
+      - name: Install nf-test
+        uses: nf-core/setup-nf-test@v1
+        with:
+          version: 0.9.5
+
+      - name: Run Tests
+        run: nf-test test --verbose
+
+  test-latest-nextflow:
+    # Runs on a schedule (and manually), not on every push/PR: this catches a future
+    # Nextflow release breaking us, without blocking normal PRs on an upstream change
+    # unrelated to them.
+    if: github.event_name == 'schedule' || github.event_name == 'workflow_dispatch'
+    runs-on: ubuntu-latest
+    timeout-minutes: 60
+
+    steps:
+      - name: Checkout
+        uses: actions/checkout@v4
+
+      - name: Setup Micromamba
+        uses: mamba-org/setup-micromamba@v1
+        with:
+          micromamba-version: '1.5.10-0' # any version from https://github.com/mamba-org/micromamba-releases
+          post-cleanup: 'all'
+
+      - name: Setup Nextflow (latest)
         uses: nf-core/setup-nextflow@v2
         with:
           version: latest
diff --git a/BENCHMARK_RESULTS.md b/BENCHMARK_RESULTS.md
new file mode 100644
index 0000000..21446e8
--- /dev/null
+++ b/BENCHMARK_RESULTS.md
@@ -0,0 +1,40 @@
+# Benchmark results: bamadap + fgumi zipper + picodup vs the current pipeline
+
+Performance changes in branch: `bamslice_zip`
+
+**Bottom line**: ~2.4x faster end-to-end on a clean dedicated node (16m/6m45s), with indistinguishable output. Correctness verified in depth (§2);
+
+## 1. What changed, and the measured impact
+
+| Change | Impact |
+| --- | --- |
+| `fastp`+`bwa`+Picard-dedup pipeline → `bamadap` + `fgumi zipper` + `picodup`, fused into single-pipe `trimAndAlign` + streamed `mergeAndPicodup` | End-to-end: **16m → 6m45s (~2.4x)** |
+| Dedup: Picard `MarkDuplicates` → `picodup`, streamed straight off `samtools merge` (no intermediate merged BAM) | **2m02s → 27s (~4.6x)**, peak RSS 8.5GB → <1GB |
+| Fixed a real `bwameth.py` bug (naive interleave-detection failed on `bamadap`'s `read/1`,`read/2` mate names, silently mis-converting bisulfite reads); fixed via `bamadap --no-mate-suffix` rather than a downstream patch | Removes a 3x slowdown *and* a silent correctness bug in the align stage |
+| `convert_methylkit_to_bed`: single-threaded `gawk` → `mawk` under `parallel --pipepart` | **7m53s → 35s (~14x)** — biggest single bottleneck in either pipeline; benefits `master` too |
+| `tasmanian` 1.x (single-threaded, capped at 2M reads, errored on some real reads) → `tasmanian-mismatch` 2.x (parallel, full library) | **3m42s → 17.5s (~13x), on more data** |
+| `fastqc` → `falco` | 57.8s → 30.8s (~2x) |
+| Fixed `gc_bias` (silently broken: `CollectGcBiasMetrics` requires an `Rscript` on PATH even when no chart is produced; `picard-slim` excludes R) | No-op `Rscript` shim; restores a previously-silent-failing QC step |
+| Right-sized cpu reservations for QC steps that don't read `task.cpus` (new `single_threaded_qc` label, fixed at 2 cpus instead of scaling with `--max_cpus`) | Frees queue slots on shared executors; no effect on single-task time |
+| Kept `bwameth --read-group` (populated from the uBAM's first `@RG`) instead of dropping it as originally planned; `zipper` still fixes the per-read `RG:Z:` tag | Avoids `picodup` mislabeling every metric row "Unknown Library" |
+| Fixed `bamadap`'s EL8 build (glibc-2.34-only symbols, `target-cpu=native` SIGILL'd on other CPUs) | Portability now solved at deploy time, not build time: Capistrano (`capistrano-rust-buildcache`) compiles a `-C target-cpu=native` binary per distinct machine type on first run and caches it, rather than shipping one generic binary |
+
+Chunk-size sweep on the real SGE cluster (18/37/55/90MB): smaller chunks buy wall-clock speed at
+the cost of more aggregate CPU-hours (7m14s/7.1 CPU-h at 18MB vs. 10m18s/4.4 CPU-h at 90MB); no
+change made to the 37MB default — it's a reasonable middle ground, not a correctness question.
+
+## 2. Correctness (final `.md.bam`, same test uBAM)
+
+| | Baseline | New | Δ |
+| --- | --- | --- | --- |
+| Total reads | 10,017,473 | 10,017,757 | +0.003% |
+| Duplicates | 1,362,823 | 1,362,959 | +0.01% |
+| PERCENT_DUPLICATION | 0.136415 | 0.136426 | matches to 4th decimal |
+| CpG methylation (Pearson r) | — | **0.999998** | 5 sites (0.0001%) differ by >1pp |
+
+Residual differences trace to expected causes (`bamadap` vs `fastp` trimming, corrected
+per-read `RG` moving optical-duplicate grouping, each dedup tool's own library-size extrapolation
+formula) — no read loss, no regression. `ngs-aggregate_results` compatibility for
+`tasmanian-mismatch` 2.x's new output format was verified against the real deployed parser
+(PR #932, merged and deployed 2026-09-07) and an end-to-end real (non-stubbed) aggregation run
+succeeded.
diff --git a/README.md b/README.md
index d6107a2..bfdd4b3 100644
--- a/README.md
+++ b/README.md
@@ -106,7 +106,7 @@ Modify the conf/references.config file to specify your genome files
 ## Reference Genomes
 Pre-built reference genomes with methylation spike-in controls + CpG Island Annotation files:
 - **T2T CHM13**: [T2T_chm13v2.0+  meth_controls](https://neb-em-seq-sra.s3.us-east-1.amazonaws.com/T2T_chm13v2.0%2Bmeth_controls.fa.gz) [CpG Islands](https://neb-em-seq-sra.s3.us-east-1.amazonaws.com/human_t2t_cpg_islands.gtf)
-- **GRCh38**: [GRCh39 + meth controls](https://neb-em-seq-sra.s3.us-east-1.amazonaws.com/grch38_core%2Bmeth_controls.fa.gz) [CpG Islands](https://neb-em-seq-sra.s3.us-east-1.amazonaws.com/human_grch38_cpg_islands.gtf)
+- **GRCh38**: [GRCh38 + meth controls](https://neb-em-seq-sra.s3.us-east-1.amazonaws.com/grch38_core%2Bmeth_controls.fa.gz) [CpG Islands](https://neb-em-seq-sra.s3.us-east-1.amazonaws.com/human_grch38_cpg_islands.gtf)
 - **GRCm39**: [GRCm39 + meth_controls](https://neb-em-seq-sra.s3.us-east-1.amazonaws.com/grcm39%2Bmeth_controls.fa.gz) [CpG Islands](https://neb-em-seq-sra.s3.us-east-1.amazonaws.com/grcm39_cpg_islands.gtf)
 - Create your own reference by appending the [control sequences](assets/methylation_controls.fa) to your preferred genome fasta (e.g. `cat genome.fa methylation_controls.fa > genome+methylation_controls.fa`)
    | Sequence | Methylation State                                | Purpose |
@@ -157,6 +157,6 @@ nf-test test --updateSnapshot
 ```
 
 ## Upgrade
-As of July, 2026, Nextflow v24 or older is not supported anymore. 
-If using Nextlfow version 24 is your only option, 
-please uncomment `nextflow.preview.topic = true` as the top line in *main.nf* 
+This pipeline requires Nextflow >=25.04, where topic channels (used throughout for
+version reporting) are a stable feature rather than a preview one. `main.nf` checks
+this at startup and fails immediately with a clear message on older versions. 
diff --git a/conf/base.config b/conf/base.config
index 468ec99..d34447c 100644
--- a/conf/base.config
+++ b/conf/base.config
@@ -23,6 +23,12 @@ process {
         cpus   = { params.max_cpus ?: 8 }
     }
 
+    withLabel: dedup {
+        // Deliberately not capped by params.max_cpus: this label exists so the dedup stage
+        // can be tuned independently of the trim/align concurrency (see BENCHMARK_PLAN.md phase 6).
+        cpus   = 16
+    }
+
     withLabel: low_cpu {
         cpus   = 2
         memory = { params.max_memory ?: 16.GB }
@@ -38,6 +44,14 @@ process {
         memory = { params.max_memory ?: 8.GB }
     }
 
+    withLabel: single_threaded_qc {
+        // For fastqc (falco), gc_bias, insert_size_metrics, picard_metrics, idx_stats:
+        // none of them read task.cpus, so scaling with params.max_cpus like medium_cpu
+        // would only reserve idle cpu slots, starving other queued tasks on the local
+        // executor for no speed benefit.
+        cpus   = 2
+    }
+
     executor = 'local' // 'sge'. 'slurm', etc... 
     
    
diff --git a/conf/references.config b/conf/references.config
index a6a1476..d26ca63 100644
--- a/conf/references.config
+++ b/conf/references.config
@@ -1,10 +1,11 @@
 params {
     genomes = [
-        'T2T_chm13v2.0+bs_controls': [
-            bwa_index: "/mnt/galaxy/galaxyworks/tool-data/T2T_chm13v2.0+bs_controls/bwameth_index/T2T_chm13v2.0+bs_controls/T2T_chm13v2.0+bs_controls.fa",
-            genome_fa: "/mnt/galaxy/galaxyworks/tool-data/T2T_chm13v2.0+bs_controls/bwameth_index/T2T_chm13v2.0+bs_controls/T2T_chm13v2.0+bs_controls.fa",
-            genome_fai: "/mnt/galaxy/galaxyworks/tool-data/T2T_chm13v2.0+bs_controls/bwameth_index/T2T_chm13v2.0+bs_controls/T2T_chm13v2.0+bs_controls.fa.fai",
-            target_bed: "/bioinfo/ref/t2t_chm13_v2+meth_controls+m13+phix/folded_windows/T2T_chm13v2.0+meth_controls_hairpins.s18w200nogt.bed"
+        'T2T_chm13v2.0+meth_controls': [
+            bwa_index:  "/bioinfo/ref/t2t_chm13v2+meth_controls/bwameth_index/T2T_chm13v2.0+bs_controls.fa",
+            genome_fa:  "/bioinfo/ref/t2t_chm13v2+meth_controls/bwameth_index/T2T_chm13v2.0+bs_controls.fa",
+            genome_fai: "/bioinfo/ref/t2t_chm13v2+meth_controls/bwameth_index/T2T_chm13v2.0+bs_controls.fa.fai",
+            genome_dict:"/bioinfo/ref/t2t_chm13v2+meth_controls/bwameth_index/T2T_chm13v2.0+bs_controls.fa.dict",
+            target_bed: "/bioinfo/ref/t2t_chm13v2+meth_controls/folded_windows/T2T_chm13v2.0+meth_controls_hairpins.s18w200nogt.bed"
         ],
         'grcm39+meth_controls': [
             bwa_index: "/mnt/galaxy/data/genome/grcm39+meth_controls/bwameth_index/grcm39+meth_controls/grcm39+meth_controls.fa",
@@ -40,6 +41,7 @@ params {
             bwa_index: "${projectDir}/tests/fixtures/reference_files/reference.fa",
             genome_fa: "${projectDir}/tests/fixtures/reference_files/reference.fa",
             genome_fai: "${projectDir}/tests/fixtures/reference_files/reference.fa.fai",
+            genome_dict: "${projectDir}/tests/fixtures/reference_files/reference.dict",
             target_bed: "${projectDir}/tests/fixtures/target_bed/emseq_test_regions.bed"
         ]
     ]
diff --git a/conf/test.config b/conf/test.config
index 89066ec..f814f87 100644
--- a/conf/test.config
+++ b/conf/test.config
@@ -24,6 +24,10 @@ params {
     max_time   = '6.h'
 
     outputDir = "test_output"
+
+    // Not yet on bioconda; point the test profile at the local EL8 builds.
+    bamadap_bin = '/appdev/langhorst/src/bamadap/target-el8/release/bamadap'
+    picodup_bin = '/appdev/langhorst/src/picodup/target-el8/release/picodup'
     }
 
     trace.overwrite = true
diff --git a/main.nf b/main.nf
index 0b15f53..b3b461d 100644
--- a/main.nf
+++ b/main.nf
@@ -1,12 +1,14 @@
-// nextflow.preview.topic = true
+// Minimum version: topic channels are stable here, preview (and unsupported) before it.
+if (!nextflow.version.matches('>=25.04')) {
+    error "This pipeline requires Nextflow >=25.04. Found: ${nextflow.version}."
+}
 
 include { registerEmailNotifications }                        from './lib/notifications.nf'
 include { createVersionsFile }                                from './lib/versions.nf'
 include { format_ngs_agg_opts }                               from './modules/aggregate_results'
-include { fastp }                                             from './modules/fastp'
 include { mergeFastpJson }                                    from './modules/merge_fastp_json'
-include { alignReads }                                        from './modules/align_reads'
-include { mergeAndMarkDuplicates }                            from './modules/merge_and_mark_duplicates'
+include { trimAndAlign }                                      from './modules/trim_and_align'
+include { mergeAndPicodup }                                   from './modules/merge_and_picodup'
 include { methylDackel_mbias }                                from './modules/methyldackel_mbias'
 include { methylDackel_extract }                              from './modules/methyldackel_extract'
 include { extract_cytosine_report }                           from './modules/extract_cytosine_report'
@@ -54,6 +56,7 @@ workflow {
          def reference_list = params.genomes[params.genome]
          genome_fa = channel.value(reference_list.genome_fa)
          genome_fai = channel.value(reference_list.genome_fai)
+         genome_dict = channel.value(reference_list.genome_dict)
 
         // Validate reference indices exist before running
         def ref = reference_list.bwa_index
@@ -63,6 +66,9 @@ workflow {
         if (!file("${reference_list.genome_fa}.fai").exists()) {
             exit 1, "Fasta index (.fai) not found for ${reference_list.genome_fa}. Run: samtools faidx ${reference_list.genome_fa}"
         }
+        if (!reference_list.genome_dict || !file(reference_list.genome_dict).exists()) {
+            exit 1, "Sequence dictionary not found for ${reference_list.genome_fa}. Run: samtools dict ${reference_list.genome_fa} -o <ref>.dict"
+        }
 
         passed_bams = bams.filter { _library, bam -> checkFileSize(bam) }
         failed_bams = bams.filter { _library, bam -> !checkFileSize(bam) }
@@ -99,23 +105,18 @@ workflow {
             }
         }
 
-        fastp( passed_bams.combine(bam_chunks, by:0), adapter_fasta )
-        mergeFastpJson( fastp.out.fastp_json.groupTuple() )
+        trimAndAlign(
+            passed_bams.combine(bam_chunks, by:0),
+            adapter_fasta,
+            reference_list.bwa_index,
+            genome_fa,
+            genome_fai,
+            genome_dict
+        )
+        mergeFastpJson( trimAndAlign.out.fastp_json.groupTuple() )
 
-        fastq_chunks = fastp.out.trimmed_fastq.map { library, chunk_name, fq_files ->
-            def fq_list = fq_files instanceof List ? fq_files : [fq_files]
-            if (params.single_end) {
-                tuple(library, chunk_name, fq_list[0])
-            }
-            else {
-                def r1 = fq_list.find { fq -> fq.name.contains('.1.trimmed.fastq') }
-                def r2 = fq_list.find { fq -> fq.name.contains('.2.trimmed.fastq') }
-                tuple(library, chunk_name, [r1, r2])
-            }
-        }
-        alignReads( passed_bams.combine(fastq_chunks, by:0), reference_list.bwa_index )
-        mergeAndMarkDuplicates( alignReads.out.bam_files.groupTuple() )
-        md_bams = mergeAndMarkDuplicates.out.md_bams
+        mergeAndPicodup( trimAndAlign.out.bam_files.groupTuple() )
+        md_bams = mergeAndPicodup.out.md_bams
 
         ///////// Methylation Calling //////////
         methylDackel_extract( md_bams, genome_fa, genome_fai )
@@ -157,18 +158,18 @@ workflow {
         fastqc( md_bams )
         picard_metrics( md_bams, genome_fa, genome_fai )
         tasmanian( md_bams, genome_fa, genome_fai )
-        combine_nonconverted_counts( alignReads.out.nonconverted_counts.groupTuple() )
+        combine_nonconverted_counts( trimAndAlign.out.nonconverted_counts.groupTuple() )
         find_switchback_reads( md_bams, genome_fa )
 
         //////// Collect files for internal summaries //////////
         agg_opts = [
-        ['--bam', mergeAndMarkDuplicates.out.md_bams.map{ row -> tuple(row[0], row[1]) }],
-        ['--bai', mergeAndMarkDuplicates.out.md_bams.map{ row -> tuple(row[0], row[2]) }],
+        ['--bam', mergeAndPicodup.out.md_bams.map{ row -> tuple(row[0], row[1]) }],
+        ['--bai', mergeAndPicodup.out.md_bams.map{ row -> tuple(row[0], row[2]) }],
         ['--metadata_bam_file', bams],
         ['--fastp', mergeFastpJson.out.merged_json],
         ['--aln', picard_metrics.out.for_agg ],
         ['--gc', gc_bias.out.for_agg ],
-        ['--dup', mergeAndMarkDuplicates.out.log],
+        ['--dup', mergeAndPicodup.out.log],
         ['--idx_stats', idx_stats.out.for_agg],
         ['--flagstat', flagstats.out.for_agg],
         ['--fastqc', fastqc.out.for_agg],
diff --git a/modules/align_reads.nf b/modules/align_reads.nf
deleted file mode 100644
index bfa90e6..0000000
--- a/modules/align_reads.nf
+++ /dev/null
@@ -1,32 +0,0 @@
-process alignReads {
-    label 'high_cpu'
-    tag { library }
-    conda "conda-forge::python=3.10 bioconda::bwameth=0.2.7 bioconda::mark-nonconverted-reads=1.2 bioconda::samtools=1.22"
-    publishDir "${params.outputDir}/bwameth_align", mode: 'symlink'
-
-    input:
-        tuple val(library), path(bam), val(chunk_name), path(reads)
-        val(genome_fa)
-
-    output:
-        tuple val(library), path("${chunk_name}.aln.bam"), path("${chunk_name}.aln.bam.bai"), emit: bam_files
-        tuple val(library), path("${chunk_name}.nonconverted_counts.tsv"), emit: nonconverted_counts
-        tuple val("${task.process}"), val('samtools'), eval('samtools --version | head -n 1 | sed \'s/^samtools //\''), topic: versions
-        tuple val("${task.process}"), val('bwameth'), eval('bwameth.py --version | sed \'s/^bwa-meth.py //\''), topic: versions
-        tuple val("${task.process}"), val('python'), eval('python --version | sed \'s/^Python //\''), topic: versions
-
-    script:
-    def bwameth_input = params.single_end ? "${reads}" : "-p ${reads[0]} ${reads[1]}"
-    """
-    set +o pipefail
-    rg_line=\$(samtools view -H ${bam} | grep "^@RG" | sed 's/\\t/\\\\t/g' | head -n1)
-    set -o pipefail
-
-    bwameth.py -t ${Math.max(1,(task.cpus*7).intdiv(8))} --read-group "\${rg_line}" --reference ${genome_fa} ${bwameth_input} 2> "${library}.log.bwamem" \
-    | mark-nonconverted-reads.py --reference ${genome_fa} 2> "${chunk_name}.nonconverted_counts.tsv" \
-    | samtools sort -T ${params.tmp_dir}/samtools_sort_tmp -@ ${Math.max(1,task.cpus.intdiv(8))} \
-       -m ${(task.memory.toGiga()*5).intdiv(8)}G --write-index \
-       -o "${chunk_name}.aln.bam##idx##${chunk_name}.aln.bam.bai" /dev/stdin
-
-    """
-}
diff --git a/modules/convert_methylkit_to_bed.nf b/modules/convert_methylkit_to_bed.nf
index 242b3b6..5db7ac7 100644
--- a/modules/convert_methylkit_to_bed.nf
+++ b/modules/convert_methylkit_to_bed.nf
@@ -1,7 +1,7 @@
 process convert_methylkit_to_bed {
-    label 'process_single'
+    label 'cpus_8'
     tag "${library}"
-    conda "conda-forge::pigz=2.8 conda-forge::gawk=5.3.1 conda-forge::sed=4.9"
+    conda "conda-forge::parallel=20250622 bioconda::mawk=1.3.4"
 
     input:
         tuple val(library), path(cytosine_report)
@@ -12,31 +12,27 @@ process convert_methylkit_to_bed {
         tuple val(library), path("${library}.bed"), emit: methylkit_bed
 
     script:
-    // Using cytosine report as a substitute for merging 3 methylkit files. 
-    // Reports C's with no coverage, so we will filter those out
+    // Using cytosine report as a substitute for merging 3 methylkit files.
+    // Reports C's with no coverage, so we will filter those out.
+    //
+    // Rows are independent, so `parallel --pipepart` seeks into byte ranges of the
+    // file with no copy and runs mawk over each concurrently; `--keep-order` keeps
+    // the output in the sorted order `bedtools intersect -sorted` needs downstream.
     """
+    # A script file, not an inline awk program: `parallel` re-quotes the command it
+    # hands each worker, which mangles braces and \$N as its own syntax.
+    cat <<'AWK' > convert_methylkit.awk
+    BEGIN { OFS = "\t" }
     # Input: chromosome, position (1-based), strand, meth_count, unmeth_count, context, trinucleotide
-    # Output: chr, start (0-based), end (1-based), context, methylation_freq, strand
-    awk '
-        BEGIN { OFS = "\t" }
-        !( \$4==0 && \$5==0 ){
-            chromosome = \$1
-            position = \$2
-            strand = \$3
-            meth_count = \$4
-            unmeth_count = \$5
-            context = \$6
-            
-            # Calculate methylation frequency
-            total_count = meth_count + unmeth_count
-            meth_freq = (total_count > 0) ? (meth_count / total_count * 100) : 0
-            
-            # Convert to BED format (0-based start, 1-based end)
-            start = position - 1
-            end = position
-            
-            print chromosome, start, end, context, meth_freq, strand
-        }
-    ' ${cytosine_report} > "${library}.bed"
+    !( \$4==0 && \$5==0 ){
+        total_count = \$4 + \$5
+        meth_freq = (total_count > 0) ? (\$4 / total_count * 100) : 0
+        # Output: chr, start (0-based), end (1-based), context, methylation_freq, strand
+        print \$1, \$2 - 1, \$2, \$6, meth_freq, \$3
+    }
+    AWK
+
+    parallel --pipepart --keep-order -j ${task.cpus} -a ${cytosine_report} \\
+        mawk -f convert_methylkit.awk > "${library}.bed"
     """
 }
diff --git a/modules/fastp.nf b/modules/fastp.nf
deleted file mode 100644
index 10b0ccf..0000000
--- a/modules/fastp.nf
+++ /dev/null
@@ -1,38 +0,0 @@
-process fastp {
-	tag "${library}"
-    label 'low_cpu'
-    conda "bioconda::samtools=1.21 bioconda::fastp=1.3.6 bioconda::bamslice=0.2.1"
-    publishDir "${params.outputDir}/fastp"
-
-    input:
-        tuple val(library), path(bam), val(start_offset), val(end_offset)
-        path(adapter_fasta)
-
-    output:
-        tuple val(library), val("${library}_${start_offset}_${end_offset}"), path("${library}_${start_offset}_${end_offset}*.trimmed.fastq.gz"), emit: trimmed_fastq
-        tuple val(library), val("${library}_${start_offset}_${end_offset}"), path("${library}_${start_offset}_${end_offset}.fastp.json"), emit: fastp_json
-        tuple val("${task.process}"), val('samtools'), eval('samtools --version | head -n 1 | sed \'s/^samtools //\''), topic: versions
-        tuple val("${task.process}"), val('fastp'), eval('fastp --version 2>&1 | cut -f 2 -d " "'), topic: versions
-        tuple val("${task.process}"), val('bamslice'), eval('bamslice --version | cut -f 2 -d " "'), topic: versions
-
-    script:
-    def chunk = "${library}_${start_offset}_${end_offset}"
-    def fastp_args = params.single_end ? "--out1 ${chunk}.1.trimmed.fastq.gz" : "--interleaved_in --out1 ${chunk}.1.trimmed.fastq.gz --out2 ${chunk}.2.trimmed.fastq.gz"
-    """
-    set +o pipefail
-    inst_name=\$(samtools view ${bam} | head -n 1 | cut -d ":" -f 1)
-    set -o pipefail
-
-    trim_polyg=\$(echo "\${inst_name}" | awk '{if (\$1~/^A0|^NB|^NS|^VH/) {print "--trim_poly_g"} else {print ""}}')
-    echo \${trim_polyg} | awk '{ if (length(\$1)>0) { print "2-color instrument: poly-g trim mode on" } }'
-
-    bamslice --input ${bam} --start-offset ${start_offset} --end-offset ${end_offset} | \\
-    fastp --stdin \\
-                    -l 2 -Q \${trim_polyg} \\
-                    --thread 1 \\
-                    --adapter_fasta ${adapter_fasta} \\
-                    --overrepresentation_analysis \\
-                    -j "${chunk}.fastp.json" \\
-                    ${fastp_args}
-    """
-}
diff --git a/modules/fastqc.nf b/modules/fastqc.nf
index f405b9b..c26c07d 100644
--- a/modules/fastqc.nf
+++ b/modules/fastqc.nf
@@ -1,7 +1,7 @@
 process fastqc {
-    label 'medium_cpu'
+    label 'single_threaded_qc'
     tag { library }
-    conda "bioconda::fastqc=0.11.8"
+    conda "bioconda::falco=1.0.0 conda-forge::zip=3.0"
     publishDir "${params.outputDir}/stats/fastqc"
 
     input:
@@ -10,10 +10,20 @@ process fastqc {
     output:
         tuple val(library), path('*_fastqc.zip'), emit: for_agg
         tuple val(library), path('*_fastqc.html'), emit: html
-        tuple val("${task.process}"), val('fastqc'), eval('fastqc --version | cut -f 2 -d " "'), topic: versions
+        tuple val("${task.process}"), val('falco'), eval('falco --version | cut -f 2 -d " "'), topic: versions
 
     script:
+    // falco's own output is flat (fastqc_data.txt, fastqc_report.html, summary.txt;
+    // no per-sample name or archive). Repackage into a <prefix>_fastqc.zip containing
+    // a <prefix>_fastqc/ directory — the layout MultiQC's fastqc module and
+    // aggregate_results.nf's `unzip *fastqc.zip` + "fastqc/fastqc_data.txt"
+    // substitution both expect. <prefix> must be the BAM filename minus ".bam"
+    // (bam.baseName), not the library tag, to match what FastQC itself would name it.
     """
-    fastqc -f bam ${bam}
+    falco -f bam ${bam}
+    mkdir ${bam.baseName}_fastqc
+    mv fastqc_data.txt summary.txt ${bam.baseName}_fastqc/
+    zip -rq ${bam.baseName}_fastqc.zip ${bam.baseName}_fastqc
+    mv fastqc_report.html ${bam.baseName}_fastqc.html
     """
 }
diff --git a/modules/gc_bias.nf b/modules/gc_bias.nf
index 783f477..cbf89f8 100644
--- a/modules/gc_bias.nf
+++ b/modules/gc_bias.nf
@@ -1,8 +1,8 @@
 
 process gc_bias {
-    label 'medium_cpu'
+    label 'single_threaded_qc'
     tag { library }
-    conda "bioconda::picard=3.3.0 bioconda::samtools=1.22"
+    conda "bioconda::picard-slim=3.5.0 bioconda::samtools=1.22"
     publishDir "${params.outputDir}/stats/gc_bias"
 
     input:
@@ -15,7 +15,14 @@ process gc_bias {
         tuple val("${task.process}"), val('picard'), eval('picard CollectGcBiasMetrics --version 2>&1 | cut -f 2 -d ":"'), topic: versions
 
     script:
+    // CollectGcBiasMetrics checks for an Rscript on PATH even with --CHART /dev/null;
+    // picard-slim excludes R, so shim a no-op Rscript rather than add a real R dependency.
     """
+    mkdir -p fakebin
+    printf '#!/bin/sh\\nexit 0\\n' > fakebin/Rscript
+    chmod +x fakebin/Rscript
+    export PATH="\$PWD/fakebin:\$PATH"
+
     samtools view -H ${bam} | grep "^@SQ" \
     | grep -v "plasmid_puc19\\|phage_lambda\\|phage_Xp12\\|phage_T4\\|EBV\\|chrM" \
     | awk -F":|\\t" '{print \$3"\\t"0"\\t"\$5}' > include_regions.bed
diff --git a/modules/idx_stats.nf b/modules/idx_stats.nf
index fcd47c0..fb631c2 100644
--- a/modules/idx_stats.nf
+++ b/modules/idx_stats.nf
@@ -1,5 +1,5 @@
 process idx_stats {
-    label 'medium_cpu'
+    label 'single_threaded_qc'
     tag { library }
     conda "bioconda::samtools=1.22"
     publishDir "${params.outputDir}/stats/idxstats"
diff --git a/modules/insert_size_metrics.nf b/modules/insert_size_metrics.nf
index c9259d5..93f4407 100644
--- a/modules/insert_size_metrics.nf
+++ b/modules/insert_size_metrics.nf
@@ -1,5 +1,5 @@
 process insert_size_metrics {
-    label 'medium_cpu'
+    label 'single_threaded_qc'
     tag { library }
     conda "bioconda::picard=3.3.0 bioconda::samtools=1.22"
     publishDir "${params.outputDir}/stats/insert_size"
diff --git a/modules/merge_and_mark_duplicates.nf b/modules/merge_and_mark_duplicates.nf
deleted file mode 100644
index c9f7009..0000000
--- a/modules/merge_and_mark_duplicates.nf
+++ /dev/null
@@ -1,50 +0,0 @@
-process mergeAndMarkDuplicates {
-    label 'cpus_8'
-    tag { library }
-    publishDir "${params.outputDir}/markduped_bams", mode: 'copy', pattern: '*.md.{bam,bai}'
-    publishDir "${params.outputDir}/stats/markdups", mode: 'copy', pattern: '*log'
-    conda "bioconda::picard=3.3.0 bioconda::samtools=1.22"
-    memory {
-    try {
-        def totalFileSize = bams.collect { bam_file -> bam_file.size() }.sum() / (1024 * 1024 * 1024)
-        def calculatedMemory = totalFileSize < 2 ? '32.GB' : totalFileSize < 8 ? '64.GB' : '128.GB'
-        return params.max_memory ?: calculatedMemory
-    }
-    catch (Exception _e) {
-        return params.max_memory ?: '64.GB'
-    }
-}
-
-    input:
-        tuple val(library), path(bams), path(bais)
-    output:
-        tuple val(library), path("${library}.md.bam"), path("${library}.md.bai"), emit: md_bams
-        tuple val(library), path('*.markdups_log'), emit: log
-        tuple val("${task.process}"), val('samtools'), eval('samtools --version | head -n 1 | sed \'s/^samtools //\''), topic: versions
-        tuple val("${task.process}"), val('picard'), eval('picard MarkDuplicates --version 2>&1 | cut -f 2 -d ":"'), topic: versions
-
-    script:
-    """
-    set +o pipefail
-    inst_name=\$(samtools view ${bams[0]} | head -n1 | cut -d ":" -f1);
-    set -o pipefail
-
-    optical_distance=\$(echo \${inst_name} | awk '{if (\$1~/^M0|^NS|^NB/) {print 100} else {print 2500}}')
-
-    samtools merge --threads ${task.cpus} ${library}.bam ${bams}
-
-    picard -Xmx${task.memory.toGiga().intdiv(2)}g MarkDuplicates \
-        --TAGGING_POLICY All \
-        --OPTICAL_DUPLICATE_PIXEL_DISTANCE \${optical_distance} \
-        --TMP_DIR ${params.tmp_dir} \
-        --CREATE_INDEX true \
-        --MAX_RECORDS_IN_RAM 5000000 \
-        --BARCODE_TAG "RX" \
-        --ASSUME_SORT_ORDER coordinate \
-        --VALIDATION_STRINGENCY SILENT \
-        --ADD_PG_TAG_TO_READS false \
-        -I ${library}.bam \
-        -O ${library}.md.bam \
-        -M ${library}.markdups_log
-    """
-}
diff --git a/modules/merge_and_picodup.nf b/modules/merge_and_picodup.nf
new file mode 100644
index 0000000..92ba103
--- /dev/null
+++ b/modules/merge_and_picodup.nf
@@ -0,0 +1,42 @@
+process mergeAndPicodup {
+    label 'dedup'
+    tag { library }
+    publishDir "${params.outputDir}/markduped_bams", mode: 'copy', pattern: '*.md.{bam,bai}'
+    publishDir "${params.outputDir}/stats/markdups", mode: 'copy', pattern: '*log'
+    conda "bioconda::samtools=1.22"
+    memory { params.max_memory ?: 16.GB }
+
+    input:
+        tuple val(library), path(bams)
+    output:
+        tuple val(library), path("${library}.md.bam"), path("${library}.md.bai"), emit: md_bams
+        tuple val(library), path('*.markdups_log'), emit: log
+        tuple val("${task.process}"), val('samtools'), eval('samtools --version | head -n 1 | sed \'s/^samtools //\''), topic: versions
+        tuple val("${task.process}"), val('picodup'), eval("${params.picodup_bin} --version | cut -f 2 -d ' '"), topic: versions
+
+    script:
+    def merge_cpus = Math.min(2, task.cpus)
+    def dedup_cpus = Math.max(1, task.cpus - merge_cpus)
+    def optical_distance_line = params.picodup_optical_distance != null
+        ? "optical_distance=${params.picodup_optical_distance}"
+        : 'optical_distance=$(echo ${inst_name} | awk \'{if ($1~/^M0|^NS|^NB/) {print 100} else {print 2500}}\')'
+    """
+    set +o pipefail
+    inst_name=\$(samtools view ${bams[0]} | head -n1 | cut -d ":" -f1)
+    set -o pipefail
+
+    ${optical_distance_line}
+
+    samtools merge --threads ${merge_cpus} -O bam -o - ${bams} \\
+    | ${params.picodup_bin} \\
+        --input /dev/stdin \\
+        --output ${library}.md.bam \\
+        --metrics ${library}.markdups_log \\
+        --threads ${dedup_cpus} \\
+        --optical-model ${params.picodup_optical_model} \\
+        --optical-distance \${optical_distance} \\
+        --tagging-policy All
+
+    samtools index -@ ${task.cpus} ${library}.md.bam ${library}.md.bai
+    """
+}
diff --git a/modules/methyldackel_mbias.nf b/modules/methyldackel_mbias.nf
index 2828b57..e91f314 100644
--- a/modules/methyldackel_mbias.nf
+++ b/modules/methyldackel_mbias.nf
@@ -11,7 +11,7 @@ process methylDackel_mbias {
         val(genome_fai)
 
     output:
-        path('*.svg'), emit: mbias_output_svg
+        path('*.svg'), optional: true, emit: mbias_output_svg
         path('*.tsv'), emit: mbias_output_tsv
         tuple val(library), path("${library}.combined_mbias.tsv"), emit: for_agg
         tuple val("${task.process}"), val('samtools'), eval('samtools --version | head -n 1 | sed \'s/^samtools //\''), topic: versions
@@ -19,6 +19,7 @@ process methylDackel_mbias {
 
     script:
     """
+    shopt -s nullglob
     echo -e "chr\tcontext\tstrand\tRead\tPosition\tnMethylated\tnUnmethylated\tnMethylated(+dups)\tnUnmethylated(+dups)" > ${library}.combined_mbias.tsv
     chrs=(`samtools view -H "${md_bam}" | grep @SQ | cut -f 2 | sed 's/SN://'| grep -v _random | grep -v chrUn | sed 's/|/\\|/'`)
 
diff --git a/modules/picard_metrics.nf b/modules/picard_metrics.nf
index ad64229..da72951 100644
--- a/modules/picard_metrics.nf
+++ b/modules/picard_metrics.nf
@@ -1,5 +1,5 @@
 process picard_metrics {
-    label 'medium_cpu'
+    label 'single_threaded_qc'
     tag { library }
     conda "bioconda::picard=3.3.0 bioconda::samtools=1.22"
     publishDir "${params.outputDir}/stats/picard_alignment_metrics"
diff --git a/modules/tasmanian.nf b/modules/tasmanian.nf
index 9441c36..09e70cf 100644
--- a/modules/tasmanian.nf
+++ b/modules/tasmanian.nf
@@ -2,11 +2,7 @@ process tasmanian {
     label 'medium_cpu'
     tag { library }
     publishDir "${params.outputDir}/stats/tasmanian"
-    conda "bioconda::samtools=1.22 bioconda::tasmanian-mismatch=1.0.9"
-
-    errorStrategy { task.attempt <= 1 ? 'retry' : 'terminate' }
-    maxRetries 1
-    memory { task.attempt > 1 ? '16 GB' : '8 GB' }
+    conda "bioconda::tasmanian-mismatch=2.0.5"
 
     input:
         tuple val(library), path(bam), path(bai)
@@ -15,14 +11,11 @@ process tasmanian {
 
     output:
         tuple val(library), path("${library}.tasmanian.csv"), emit: for_agg
-        tuple val("${task.process}"), val('samtools'), eval('samtools --version | head -n 1 | sed \'s/^samtools //\''), topic: versions
-        tuple val("${task.process}"), val('tasmanian'), val('*should be* 1.0.9'), topic: versions
+        tuple val("${task.process}"), val('tasmanian-mismatch'), eval('tasmanian-mismatch --version | cut -f 2 -d " "'), topic: versions
 
     script:
     """
-    set +e
-    set +o pipefail
-    samtools view -q 30 -F 3840 ${bam} | head -n 2000000 | run_tasmanian -r ${genome_fa} > ${library}.tasmanian.csv
+    tasmanian-mismatch --min-base-quality 20 --min-map-quality 30 \\
+        -F 3840 --threads ${task.cpus} -o ${library}.tasmanian.csv ${bam} ${genome_fa}
     """
-
 }
diff --git a/modules/trim_and_align.nf b/modules/trim_and_align.nf
new file mode 100644
index 0000000..0df3b20
--- /dev/null
+++ b/modules/trim_and_align.nf
@@ -0,0 +1,59 @@
+process trimAndAlign {
+    label 'high_cpu'
+    tag { library }
+    conda "conda-forge::python=3.10 bioconda::bwameth=0.2.7 bioconda::mark-nonconverted-reads=1.2 bioconda::samtools=1.22 bioconda::bamslice=0.2.1 bioconda::fgumi=0.7.0"
+    publishDir "${params.outputDir}/bwameth_align", mode: 'symlink'
+
+    input:
+        tuple val(library), path(bam), val(start_offset), val(end_offset)
+        path(adapter_fasta)
+        val(bwa_index)
+        path(genome_fa)
+        path(genome_fai)
+        path(genome_dict)
+
+    output:
+        tuple val(library), path("${chunk}.aln.bam"), emit: bam_files
+        tuple val(library), val(chunk), path("${chunk}.fastp.json"), emit: fastp_json
+        tuple val(library), path("${chunk}.nonconverted_counts.tsv"), emit: nonconverted_counts
+        tuple val("${task.process}"), val('samtools'), eval('samtools --version | head -n 1 | sed \'s/^samtools //\''), topic: versions
+        tuple val("${task.process}"), val('bwameth'), eval("bwameth.py --version | sed 's/^bwa-meth.py //'"), topic: versions
+        tuple val("${task.process}"), val('python'), eval('python --version | sed \'s/^Python //\''), topic: versions
+        tuple val("${task.process}"), val('bamslice'), eval('bamslice --version | cut -f 2 -d " "'), topic: versions
+        tuple val("${task.process}"), val('fgumi'), eval('fgumi --version | cut -f 2 -d " "'), topic: versions
+        tuple val("${task.process}"), val('bamadap'), eval("${params.bamadap_bin} --version | cut -f 2 -d ' '"), topic: versions
+
+    script:
+    chunk = "${library}_${start_offset}_${end_offset}"
+    def trim_cpus  = 4
+    def align_cpus = Math.max(1, (task.cpus * 7).intdiv(8))
+    def zip_cpus   = 2
+    def sort_cpus  = Math.max(1, task.cpus.intdiv(8))
+    def bwameth_pairing = params.single_end ? '' : '-p'
+    def slice_path = "${params.tmp_dir}/${chunk}.slice.bam"
+    """
+    set +o pipefail
+    inst_name=\$(samtools view ${bam} | head -n 1 | cut -d ":" -f 1)
+    rg_line=\$(samtools view -H ${bam} | grep "^@RG" | sed 's/\\t/\\\\t/g' | head -n1)
+    set -o pipefail
+    trim_polyg=\$(echo "\${inst_name}" | awk '{if (\$1~/^A0|^NB|^NS|^VH|^LH/) {print "--trim-poly-g"} else {print ""}}')
+
+    # we write this to a temporary file on local storage so 
+    # that both bamadap and fgumi can access it simply
+    bamslice --input ${bam} --start-offset ${start_offset} --end-offset ${end_offset} \\
+        --format bam --output ${slice_path}
+
+    ${params.bamadap_bin} --input ${slice_path} --format fastq --no-mate-suffix \\
+        -l 2 --disable-quality-filtering \\
+        --adapter-fasta ${adapter_fasta} \${trim_polyg} \\
+        --threads ${trim_cpus} --json "${chunk}.fastp.json" \\
+    | bwameth.py -t ${align_cpus} --read-group "\${rg_line}" --reference ${bwa_index} ${bwameth_pairing} - 2> "${library}.log.bwamem" \\
+    | mark-nonconverted-reads.py --reference ${bwa_index} 2> "${chunk}.nonconverted_counts.tsv" \\
+    | fgumi zipper --unmapped ${slice_path} --reference ${genome_fa} \\
+        --exclude-missing-reads true --threads ${zip_cpus} --compression-level 0 \\
+    | samtools sort -T ${params.tmp_dir}/samtools_sort_tmp -@ ${sort_cpus} \\
+        -m ${(task.memory.toGiga()*5).intdiv(8)}G -o "${chunk}.aln.bam" /dev/stdin
+
+    rm -f ${slice_path}
+    """
+}
diff --git a/nextflow.config b/nextflow.config
index b83c626..5b140d9 100644
--- a/nextflow.config
+++ b/nextflow.config
@@ -26,8 +26,20 @@ params {
     // null means no cap — per-process defaults in conf/base.config apply.
     max_memory                = null
 
+    // Optional cpu cap applied to labeled processes (e.g. --max_cpus 8).
+    // null means no cap — per-process defaults in conf/base.config apply.
+    max_cpus                  = null
+
+    // Binaries not yet packaged for bioconda; deployed to production via Capistrano
+    // (nebiolabs/bamadap, nebiolabs/picodup). Override with a different absolute path
+    // (e.g. conf/test.config) until bioconda packaging lands.
+    bamadap_bin               = '/mnt/bioinfo/prg/bamadap/current/bin/bamadap'
+    picodup_bin               = '/mnt/bioinfo/prg/picodup/current/bin/picodup'
+
+    picodup_optical_model     = 'ellipse'
+    picodup_optical_distance  = null   // null => derive from instrument name, as Picard path does
+
     // NEB only
-    path_to_ngs_agg           = null
     workflow_name_modifier    = null
 
 }
@@ -85,12 +97,6 @@ env {
     PYTHONNOUSERSITE = 1
 }
 
-executor {
-    name = 'sge'
-    queueSize = 10000
-    jobName = { "${task.process.tokenize(':')[-1]}" }
-}
-
 process.shell = ['/bin/bash', '-euo', 'pipefail']
 
 timeline {
diff --git a/tests/main.nf.test b/tests/main.nf.test
index 4690a8c..67ac147 100644
--- a/tests/main.nf.test
+++ b/tests/main.nf.test
@@ -74,11 +74,11 @@ nextflow_pipeline {
 
     test("EM-seq workflow uBam - single-end") {
         when {
-            params.ubam_dir = "$projectDir/tests/fixtures/ubam/"
+            params.ubam_dir = "$projectDir/tests/fixtures/ubam_single_end/"
             params.genome = 'test'
             params.email = 'foo@example.com'
             params.flowcell = "test_pipeline"
-            params.bamslice_chunk_size = 250000
+            params.bamslice_chunk_size = 300000
             params.enable_neb_agg = true
             params.single_end = true
             params.adapter_set = "test"
@@ -86,33 +86,31 @@ nextflow_pipeline {
         }
 
         then {
-            def bwameth_file_1 = bam("${launchDir}/test_output/bwameth_align/emseq-test1_0_250000.aln.bam").getStatistics()
-            def bwameth_file_2 = bam("${launchDir}/test_output/bwameth_align/emseq-test1_250000_421605.aln.bam").getStatistics()
-            def fastqc = path("${launchDir}/test_output/stats/fastqc/emseq-test1.md_fastqc.html").exists()
-            def fastp = path("${launchDir}/test_output/fastp/emseq-test1.fastp.json").text.tokenize('\n')[1..33]
-            def markdup = bam("${launchDir}/test_output/markduped_bams/emseq-test1.md.bam").getStatistics()
-            def markdup_log = path("${launchDir}/test_output/stats/markdups/emseq-test1.markdups_log").text.tokenize('\n')[5..6]
-            def idxstats = path("${launchDir}/test_output/stats/idxstats/emseq-test1.idxstat").text.tokenize('\n')
-            def flagstats = path("${launchDir}/test_output/stats/flagstats/emseq-test1.flagstat").text.tokenize('\n')
-            def gc_bias = path("${launchDir}/test_output/stats/gc_bias/emseq-test1.gc_metrics").text.tokenize('\n')[5..-1]
-            def alignment_metrics = path("${launchDir}/test_output/stats/picard_alignment_metrics/emseq-test1.alignment_summary_metrics.txt").text.tokenize('\n')[5..8]
-            def methyldackel_extract = path("${launchDir}/test_output/methylDackelExtracts/emseq-test1_CpG.methylKit.gz").md5
-            def mbias = path("${launchDir}/test_output/methylDackelExtracts/mbias/emseq-test1.combined_mbias.tsv").md5
-            def nonconverted = path("${launchDir}/test_output/bwameth_align/emseq-test1_0_250000.nonconverted_counts.tsv").text.tokenize('\n')
-            def nonconverted_combined = path("${launchDir}/test_output/stats/nonconverted_counts/emseq-test1.nonconverted_counts.for_agg.tsv").text.tokenize('\n')
+            def bwameth_file_1 = bam("${launchDir}/test_output/bwameth_align/emseq-test1.ds.1_0_257200.aln.bam").getStatistics()
+            def fastqc = path("${launchDir}/test_output/stats/fastqc/emseq-test1.ds.1.md_fastqc.html").exists()
+            def fastp = path("${launchDir}/test_output/fastp/emseq-test1.ds.1.fastp.json").text.tokenize('\n')[1..33]
+            def markdup = bam("${launchDir}/test_output/markduped_bams/emseq-test1.ds.1.md.bam").getStatistics()
+            def markdup_log = path("${launchDir}/test_output/stats/markdups/emseq-test1.ds.1.markdups_log").text.tokenize('\n')[5..6]
+            def idxstats = path("${launchDir}/test_output/stats/idxstats/emseq-test1.ds.1.idxstat").text.tokenize('\n')
+            def flagstats = path("${launchDir}/test_output/stats/flagstats/emseq-test1.ds.1.flagstat").text.tokenize('\n')
+            def gc_bias = path("${launchDir}/test_output/stats/gc_bias/emseq-test1.ds.1.gc_metrics").text.tokenize('\n')[5..-1]
+            def alignment_metrics = path("${launchDir}/test_output/stats/picard_alignment_metrics/emseq-test1.ds.1.alignment_summary_metrics.txt").text.tokenize('\n')[5..8]
+            def methyldackel_extract = path("${launchDir}/test_output/methylDackelExtracts/emseq-test1.ds.1_CpG.methylKit.gz").md5
+            def mbias = path("${launchDir}/test_output/methylDackelExtracts/mbias/emseq-test1.ds.1.combined_mbias.tsv").md5
+            def nonconverted = path("${launchDir}/test_output/bwameth_align/emseq-test1.ds.1_0_257200.nonconverted_counts.tsv").text.tokenize('\n')
+            def nonconverted_combined = path("${launchDir}/test_output/stats/nonconverted_counts/emseq-test1.ds.1.nonconverted_counts.for_agg.tsv").text.tokenize('\n')
             def combined_summaries = path("${launchDir}/test_output/methylKit_intersections/positional_summaries_combined.tsv").exists()
-            def positional_summary = path("${launchDir}/test_output/methylKit_intersections/emseq-test1_vs_emseq_test_regions_positional_summary.tsv").text.tokenize('\n')
-            def methylkit_intersections = path("${launchDir}/test_output/methylKit_intersections/emseq-test2_vs_emseq_test_regions_intersections.tsv").text.tokenize('\n')
-            def region_summary = path("${launchDir}/test_output/methylKit_intersections/emseq-test2_vs_emseq_test_regions_region_summary.tsv").text.tokenize('\n')
-            def tasmanian = path("${launchDir}/test_output/stats/tasmanian/emseq-test1.tasmanian.csv").exists()
+            def positional_summary = path("${launchDir}/test_output/methylKit_intersections/emseq-test1.ds.1_vs_emseq_test_regions_positional_summary.tsv").text.tokenize('\n')
+            def methylkit_intersections = path("${launchDir}/test_output/methylKit_intersections/emseq-test2.ds.1_vs_emseq_test_regions_intersections.tsv").text.tokenize('\n')
+            def region_summary = path("${launchDir}/test_output/methylKit_intersections/emseq-test2.ds.1_vs_emseq_test_regions_region_summary.tsv").text.tokenize('\n')
+            def tasmanian = path("${launchDir}/test_output/stats/tasmanian/emseq-test1.ds.1.tasmanian.csv").exists()
             def multiqc = path("${launchDir}/test_output/EM-seq-Alignment-Summary-test_pipeline_multiqc_report.html").exists()
-            def ngs_agg = path("${launchDir}/test_output/aggregate_results/emseq-test1.arguments.txt").text.tokenize('\n')
+            def ngs_agg = path("${launchDir}/test_output/aggregate_results/emseq-test1.ds.1.arguments.txt").text.tokenize('\n')
 
             assertAll(
                 { assert workflow.success },
                 { assert snapshot(workflow.trace,
                     ["bwameth_file_1", bwameth_file_1],
-                    ["bwameth_file_2", bwameth_file_2],
                     ["fastqc", fastqc],
                     ["fastp", fastp],
                     ["markdup", markdup],
diff --git a/tests/main.nf.test.snap b/tests/main.nf.test.snap
index fbf1058..c199cde 100644
--- a/tests/main.nf.test.snap
+++ b/tests/main.nf.test.snap
@@ -2,32 +2,20 @@
     "EM-seq workflow uBam - single-end": {
         "content": [
             {
-                "tasksCount": 47,
+                "tasksCount": 41,
                 "tasksFailed": 0,
-                "tasksSucceeded": 47
+                "tasksSucceeded": 41
             },
             [
                 "bwameth_file_1",
                 {
-                    "maxReadLength": 76,
-                    "minReadLength": 44,
-                    "meanReadLength": 75,
-                    "maxQuality": 60,
-                    "minQuality": 0,
-                    "meanQuality": 10,
-                    "readCount": 702
-                }
-            ],
-            [
-                "bwameth_file_2",
-                {
-                    "maxReadLength": 76,
-                    "minReadLength": 52,
-                    "meanReadLength": 75,
+                    "maxReadLength": 150,
+                    "minReadLength": 53,
+                    "meanReadLength": 146,
                     "maxQuality": 60,
                     "minQuality": 0,
-                    "meanQuality": 8,
-                    "readCount": 399
+                    "meanQuality": 41,
+                    "readCount": 763
                 }
             ],
             [
@@ -38,35 +26,35 @@
                 "fastp",
                 [
                     "\t\"summary\": {",
-                    "\t\t\"fastp_version\": \"1.3.6\",",
-                    "\t\t\"sequencing\": \"single end (76 cycles)\",",
+                    "\t\t\"fastp_version\": \"0.1.0\",",
+                    "\t\t\"sequencing\": \"single end (150 cycles)\",",
                     "\t\t\"before_filtering\": {",
-                    "\t\t\t\"total_reads\": 9534,",
-                    "\t\t\t\"total_bases\": 724584,",
-                    "\t\t\t\"q20_bases\": 681052,",
-                    "\t\t\t\"q30_bases\": 662196,",
-                    "\t\t\t\"q20_rate\": 0.939921,",
-                    "\t\t\t\"q30_rate\": 0.913898,",
-                    "\t\t\t\"read1_mean_length\": 76,",
-                    "\t\t\t\"gc_content\": 0.218667",
+                    "\t\t\t\"total_reads\": 2500,",
+                    "\t\t\t\"total_bases\": 375000,",
+                    "\t\t\t\"q20_bases\": 368284,",
+                    "\t\t\t\"q30_bases\": 356222,",
+                    "\t\t\t\"q20_rate\": 0.982091,",
+                    "\t\t\t\"q30_rate\": 0.949925,",
+                    "\t\t\t\"read1_mean_length\": 150,",
+                    "\t\t\t\"gc_content\": 0.214909",
                     "\t\t},",
                     "\t\t\"after_filtering\": {",
-                    "\t\t\t\"total_reads\": 9530,",
-                    "\t\t\t\"total_bases\": 721079,",
-                    "\t\t\t\"q20_bases\": 678095,",
-                    "\t\t\t\"q30_bases\": 659406,",
-                    "\t\t\t\"q20_rate\": 0.940389,",
-                    "\t\t\t\"q30_rate\": 0.914471,",
-                    "\t\t\t\"read1_mean_length\": 75,",
-                    "\t\t\t\"gc_content\": 0.216831",
+                    "\t\t\t\"total_reads\": 2500,",
+                    "\t\t\t\"total_bases\": 371387,",
+                    "\t\t\t\"q20_bases\": 364731,",
+                    "\t\t\t\"q30_bases\": 352753,",
+                    "\t\t\t\"q20_rate\": 0.982078,",
+                    "\t\t\t\"q30_rate\": 0.949826,",
+                    "\t\t\t\"read1_mean_length\": 148,",
+                    "\t\t\t\"gc_content\": 0.211981",
                     "\t\t}",
                     "\t},",
                     "\t\"filtering_result\": {",
-                    "\t\t\"passed_filter_reads\": 9530,",
+                    "\t\t\"passed_filter_reads\": 2500,",
                     "\t\t\"low_quality_reads\": 0,",
                     "\t\t\"too_many_N_reads\": 0,",
                     "\t\t\"adapter_dimer_reads\": 0,",
-                    "\t\t\"too_short_reads\": 4,",
+                    "\t\t\"too_short_reads\": 0,",
                     "\t\t\"too_long_reads\": 0",
                     "\t},",
                     "\t\"duplication\": {"
@@ -75,40 +63,40 @@
             [
                 "markdup",
                 {
-                    "maxReadLength": 76,
-                    "minReadLength": 44,
-                    "meanReadLength": 75,
+                    "maxReadLength": 150,
+                    "minReadLength": 53,
+                    "meanReadLength": 146,
                     "maxQuality": 60,
                     "minQuality": 0,
-                    "meanQuality": 9,
-                    "readCount": 1101
+                    "meanQuality": 41,
+                    "readCount": 763
                 }
             ],
             [
                 "markdup_log",
                 [
                     "LIBRARY\tUNPAIRED_READS_EXAMINED\tREAD_PAIRS_EXAMINED\tSECONDARY_OR_SUPPLEMENTARY_RDS\tUNMAPPED_READS\tUNPAIRED_READ_DUPLICATES\tREAD_PAIR_DUPLICATES\tREAD_PAIR_OPTICAL_DUPLICATES\tPERCENT_DUPLICATION\tESTIMATED_LIBRARY_SIZE",
-                    "Oct25_PV_10ngR3_F6\t1100\t0\t1\t0\t81\t0\t0\t0.073636\t"
+                    "emseq-test1.ds.1\t745\t0\t18\t0\t14\t0\t0\t0.018792\t"
                 ]
             ],
             [
                 "idxstats",
                 [
-                    "chr1\t284940\t1101\t0",
+                    "chr1\t284940\t763\t0",
                     "*\t0\t0\t0"
                 ]
             ],
             [
                 "flagstats",
                 [
-                    "1095 + 6 in total (QC-passed reads + QC-failed reads)",
-                    "1094 + 6 primary",
-                    "1 + 0 secondary",
+                    "763 + 0 in total (QC-passed reads + QC-failed reads)",
+                    "745 + 0 primary",
+                    "18 + 0 secondary",
                     "0 + 0 supplementary",
-                    "81 + 0 duplicates",
-                    "81 + 0 primary duplicates",
-                    "1095 + 6 mapped (100.00% : 100.00%)",
-                    "1094 + 6 primary mapped (100.00% : 100.00%)",
+                    "14 + 0 duplicates",
+                    "14 + 0 primary duplicates",
+                    "763 + 0 mapped (100.00% : N/A)",
+                    "745 + 0 primary mapped (100.00% : N/A)",
                     "0 + 0 paired in sequencing",
                     "0 + 0 read1",
                     "0 + 0 read2",
@@ -123,59 +111,59 @@
                 "gc_bias",
                 [
                     "ACCUMULATION_LEVEL\tREADS_USED\tGC\tWINDOWS\tREAD_STARTS\tMEAN_BASE_QUALITY\tNORMALIZED_COVERAGE\tERROR_BAR_WIDTH\tSAMPLE\tLIBRARY\tREAD_GROUP",
-                    "All Reads\tALL\t0\t17252\t122\t6\t1.8295\t0.165635\t\t\t",
-                    "All Reads\tALL\t1\t3503\t37\t6\t2.732585\t0.449234\t\t\t",
-                    "All Reads\tALL\t2\t5640\t47\t6\t2.155911\t0.314472\t\t\t",
-                    "All Reads\tALL\t3\t3475\t40\t6\t2.977949\t0.470855\t\t\t",
-                    "All Reads\tALL\t4\t4275\t45\t5\t2.723256\t0.405959\t\t\t",
-                    "All Reads\tALL\t5\t3480\t18\t5\t1.338152\t0.315405\t\t\t",
-                    "All Reads\tALL\t6\t3400\t23\t5\t1.750093\t0.36492\t\t\t",
-                    "All Reads\tALL\t7\t3235\t19\t6\t1.519468\t0.34859\t\t\t",
-                    "All Reads\tALL\t8\t3640\t7\t6\t0.497518\t0.188044\t\t\t",
-                    "All Reads\tALL\t9\t3412\t11\t6\t0.834057\t0.251478\t\t\t",
-                    "All Reads\tALL\t10\t3608\t12\t6\t0.860452\t0.248391\t\t\t",
-                    "All Reads\tALL\t11\t3699\t12\t5\t0.839284\t0.24228\t\t\t",
-                    "All Reads\tALL\t12\t3840\t7\t6\t0.471606\t0.17825\t\t\t",
-                    "All Reads\tALL\t13\t3874\t8\t7\t0.534248\t0.188885\t\t\t",
-                    "All Reads\tALL\t14\t3693\t15\t6\t1.05081\t0.271318\t\t\t",
-                    "All Reads\tALL\t15\t3986\t22\t7\t1.427899\t0.304429\t\t\t",
-                    "All Reads\tALL\t16\t3911\t17\t7\t1.124536\t0.27274\t\t\t",
-                    "All Reads\tALL\t17\t3778\t16\t7\t1.095646\t0.273911\t\t\t",
-                    "All Reads\tALL\t18\t3793\t25\t7\t1.705176\t0.341035\t\t\t",
-                    "All Reads\tALL\t19\t3698\t30\t7\t2.098778\t0.383183\t\t\t",
-                    "All Reads\tALL\t20\t3735\t28\t7\t1.939454\t0.366522\t\t\t",
-                    "All Reads\tALL\t21\t3759\t34\t5\t2.340015\t0.401309\t\t\t",
-                    "All Reads\tALL\t22\t3461\t35\t6\t2.616246\t0.442226\t\t\t",
-                    "All Reads\tALL\t23\t3475\t41\t7\t3.052398\t0.476705\t\t\t",
-                    "All Reads\tALL\t24\t3393\t62\t7\t4.727374\t0.600377\t\t\t",
-                    "All Reads\tALL\t25\t3365\t52\t7\t3.997886\t0.554407\t\t\t",
-                    "All Reads\tALL\t26\t3200\t43\t6\t3.476407\t0.530147\t\t\t",
-                    "All Reads\tALL\t27\t2988\t38\t8\t3.290146\t0.533732\t\t\t",
-                    "All Reads\tALL\t28\t2852\t35\t6\t3.174904\t0.536657\t\t\t",
-                    "All Reads\tALL\t29\t2805\t29\t8\t2.674713\t0.496682\t\t\t",
-                    "All Reads\tALL\t30\t2833\t10\t5\t0.913199\t0.288779\t\t\t",
-                    "All Reads\tALL\t31\t2731\t23\t7\t2.178805\t0.454312\t\t\t",
-                    "All Reads\tALL\t32\t2760\t25\t8\t2.343382\t0.468676\t\t\t",
-                    "All Reads\tALL\t33\t2776\t16\t6\t1.49112\t0.37278\t\t\t",
-                    "All Reads\tALL\t34\t2475\t15\t5\t1.567935\t0.404839\t\t\t",
-                    "All Reads\tALL\t35\t2672\t14\t6\t1.355513\t0.362276\t\t\t",
-                    "All Reads\tALL\t36\t2796\t7\t8\t0.647699\t0.244807\t\t\t",
-                    "All Reads\tALL\t37\t2850\t9\t11\t0.816977\t0.272326\t\t\t",
-                    "All Reads\tALL\t38\t2826\t11\t7\t1.007007\t0.303624\t\t\t",
-                    "All Reads\tALL\t39\t3054\t4\t10\t0.338847\t0.169423\t\t\t",
-                    "All Reads\tALL\t40\t3449\t6\t8\t0.45006\t0.183736\t\t\t",
-                    "All Reads\tALL\t41\t4012\t9\t5\t0.580355\t0.193452\t\t\t",
-                    "All Reads\tALL\t42\t4971\t7\t5\t0.364306\t0.137695\t\t\t",
-                    "All Reads\tALL\t43\t5630\t5\t6\t0.22976\t0.102752\t\t\t",
-                    "All Reads\tALL\t44\t6409\t2\t11\t0.080733\t0.057087\t\t\t",
-                    "All Reads\tALL\t45\t7297\t2\t8\t0.070908\t0.05014\t\t\t",
-                    "All Reads\tALL\t46\t8146\t2\t10\t0.063518\t0.044914\t\t\t",
-                    "All Reads\tALL\t47\t8876\t1\t8\t0.029147\t0.029147\t\t\t",
+                    "All Reads\tALL\t0\t17252\t5\t8\t0.108195\t0.048386\t\t\t",
+                    "All Reads\tALL\t1\t3503\t2\t10\t0.21314\t0.150713\t\t\t",
+                    "All Reads\tALL\t2\t5640\t2\t9\t0.132381\t0.093608\t\t\t",
+                    "All Reads\tALL\t3\t3475\t0\t0\t0\t0\t\t\t",
+                    "All Reads\tALL\t4\t4275\t2\t12\t0.17465\t0.123496\t\t\t",
+                    "All Reads\tALL\t5\t3480\t2\t0\t0.214549\t0.151709\t\t\t",
+                    "All Reads\tALL\t6\t3400\t1\t0\t0.109798\t0.109798\t\t\t",
+                    "All Reads\tALL\t7\t3235\t3\t11\t0.346196\t0.199876\t\t\t",
+                    "All Reads\tALL\t8\t3640\t7\t30\t0.717913\t0.271345\t\t\t",
+                    "All Reads\tALL\t9\t3412\t6\t17\t0.656473\t0.268004\t\t\t",
+                    "All Reads\tALL\t10\t3608\t4\t28\t0.413874\t0.206937\t\t\t",
+                    "All Reads\tALL\t11\t3699\t15\t33\t1.513847\t0.390873\t\t\t",
+                    "All Reads\tALL\t12\t3840\t20\t20\t1.944347\t0.434769\t\t\t",
+                    "All Reads\tALL\t13\t3874\t25\t17\t2.409103\t0.481821\t\t\t",
+                    "All Reads\tALL\t14\t3693\t23\t16\t2.325003\t0.484797\t\t\t",
+                    "All Reads\tALL\t15\t3986\t38\t19\t3.558945\t0.577337\t\t\t",
+                    "All Reads\tALL\t16\t3911\t25\t14\t2.386311\t0.477262\t\t\t",
+                    "All Reads\tALL\t17\t3778\t18\t14\t1.778629\t0.419227\t\t\t",
+                    "All Reads\tALL\t18\t3793\t36\t16\t3.543191\t0.590532\t\t\t",
+                    "All Reads\tALL\t19\t3698\t38\t16\t3.836115\t0.6223\t\t\t",
+                    "All Reads\tALL\t20\t3735\t37\t14\t3.698163\t0.607974\t\t\t",
+                    "All Reads\tALL\t21\t3759\t40\t14\t3.972488\t0.628105\t\t\t",
+                    "All Reads\tALL\t22\t3461\t31\t14\t3.343759\t0.600557\t\t\t",
+                    "All Reads\tALL\t23\t3475\t31\t13\t3.330288\t0.598137\t\t\t",
+                    "All Reads\tALL\t24\t3393\t27\t13\t2.970673\t0.571706\t\t\t",
+                    "All Reads\tALL\t25\t3365\t44\t12\t4.881379\t0.735896\t\t\t",
+                    "All Reads\tALL\t26\t3200\t27\t12\t3.149841\t0.606187\t\t\t",
+                    "All Reads\tALL\t27\t2988\t33\t13\t4.122952\t0.717714\t\t\t",
+                    "All Reads\tALL\t28\t2852\t37\t12\t4.843141\t0.796207\t\t\t",
+                    "All Reads\tALL\t29\t2805\t38\t13\t5.057381\t0.820415\t\t\t",
+                    "All Reads\tALL\t30\t2833\t21\t12\t2.767245\t0.603862\t\t\t",
+                    "All Reads\tALL\t31\t2731\t19\t10\t2.597208\t0.59584\t\t\t",
+                    "All Reads\tALL\t32\t2760\t18\t11\t2.43466\t0.573855\t\t\t",
+                    "All Reads\tALL\t33\t2776\t23\t13\t3.093024\t0.64494\t\t\t",
+                    "All Reads\tALL\t34\t2475\t12\t14\t1.81001\t0.522505\t\t\t",
+                    "All Reads\tALL\t35\t2672\t8\t13\t1.117708\t0.39517\t\t\t",
+                    "All Reads\tALL\t36\t2796\t6\t12\t0.801104\t0.327049\t\t\t",
+                    "All Reads\tALL\t37\t2850\t13\t11\t1.702838\t0.472282\t\t\t",
+                    "All Reads\tALL\t38\t2826\t5\t12\t0.6605\t0.295385\t\t\t",
+                    "All Reads\tALL\t39\t3054\t4\t13\t0.488952\t0.244476\t\t\t",
+                    "All Reads\tALL\t40\t3449\t0\t0\t0\t0\t\t\t",
+                    "All Reads\tALL\t41\t4012\t6\t13\t0.558297\t0.227924\t\t\t",
+                    "All Reads\tALL\t42\t4971\t5\t12\t0.375492\t0.167925\t\t\t",
+                    "All Reads\tALL\t43\t5630\t1\t13\t0.066308\t0.066308\t\t\t",
+                    "All Reads\tALL\t44\t6409\t0\t0\t0\t0\t\t\t",
+                    "All Reads\tALL\t45\t7297\t1\t12\t0.05116\t0.05116\t\t\t",
+                    "All Reads\tALL\t46\t8146\t1\t8\t0.045828\t0.045828\t\t\t",
+                    "All Reads\tALL\t47\t8876\t2\t8\t0.084118\t0.05948\t\t\t",
                     "All Reads\tALL\t48\t9213\t0\t0\t0\t0\t\t\t",
                     "All Reads\tALL\t49\t9815\t0\t0\t0\t0\t\t\t",
-                    "All Reads\tALL\t50\t10134\t1\t6\t0.025529\t0.025529\t\t\t",
-                    "All Reads\tALL\t51\t9913\t1\t3\t0.026098\t0.026098\t\t\t",
-                    "All Reads\tALL\t52\t9189\t1\t2\t0.028154\t0.028154\t\t\t",
+                    "All Reads\tALL\t50\t10134\t1\t22\t0.036838\t0.036838\t\t\t",
+                    "All Reads\tALL\t51\t9913\t0\t0\t0\t0\t\t\t",
+                    "All Reads\tALL\t52\t9189\t0\t0\t0\t0\t\t\t",
                     "All Reads\tALL\t53\t8455\t0\t0\t0\t0\t\t\t",
                     "All Reads\tALL\t54\t7312\t0\t0\t0\t0\t\t\t",
                     "All Reads\tALL\t55\t5984\t0\t0\t0\t0\t\t\t",
@@ -230,18 +218,18 @@
                 "alignment_metrics",
                 [
                     "CATEGORY\tTOTAL_READS\tPF_READS\tPCT_PF_READS\tPF_NOISE_READS\tPF_READS_ALIGNED\tPCT_PF_READS_ALIGNED\tPF_ALIGNED_BASES\tPF_HQ_ALIGNED_READS\tPF_HQ_ALIGNED_BASES\tPF_HQ_ALIGNED_Q20_BASES\tPF_HQ_MEDIAN_MISMATCHES\tPF_MISMATCH_RATE\tPF_HQ_ERROR_RATE\tPF_INDEL_RATE\tMEAN_READ_LENGTH\tSD_READ_LENGTH\tMEDIAN_READ_LENGTH\tMAD_READ_LENGTH\tMIN_READ_LENGTH\tMAX_READ_LENGTH\tMEAN_ALIGNED_READ_LENGTH\tREADS_ALIGNED_IN_PAIRS\tPCT_READS_ALIGNED_IN_PAIRS\tPF_READS_IMPROPER_PAIRS\tPCT_PF_READS_IMPROPER_PAIRS\tBAD_CYCLES\tSTRAND_BALANCE\tPCT_CHIMERAS\tPCT_ADAPTER\tPCT_SOFTCLIP\tPCT_HARDCLIP\tAVG_POS_3PRIME_SOFTCLIP_LENGTH\tSAMPLE\tLIBRARY\tREAD_GROUP",
-                    "UNPAIRED\t1100\t1094\t0.994545\t0\t1094\t1\t80799\t224\t16598\t15805\t0\t0.205196\t0.161755\t0.004616\t75.754113\t2.037807\t76\t0\t44\t76\t74.196527\t0\t0\t0\t0\t0\t0.674589\t0.004464\t0\t0.020561\t0\t18.139535\t\t\t",
+                    "UNPAIRED\t745\t745\t1\t0\t745\t1\t99162\t537\t76242\t75002\t0\t0.034304\t0.014897\t0.002219\t148.559732\t6.956237\t150\t0\t71\t150\t133.465772\t0\t0\t0\t0\t0\t0.989262\t0.018622\t0\t0.101602\t0\t49.564103\t\t\t",
                     "## HISTOGRAM\tjava.lang.Integer",
                     "READ_LENGTH\tUNPAIRED_TOTAL_LENGTH_COUNT\tUNPAIRED_ALIGNED_LENGTH_COUNT"
                 ]
             ],
             [
                 "methyldackel_extract",
-                "91eac7636f2e617927f190e3e884b8c9"
+                "55ab0743f01e4d66921bacd7dd3ef411"
             ],
             [
                 "mbias",
-                "824e46917dfa852f1f3ed83571008eef"
+                "20c46a85d89d9fa53ed671a1372a7299"
             ],
             [
                 "nonconverted",
@@ -252,15 +240,17 @@
             [
                 "nonconverted_combined",
                 [
-                    "emseq-test1\tchr1\t0"
+                    "emseq-test1.ds.1\tchr1\t0"
                 ]
             ],
             [
                 "positional_summary",
                 [
                     "methylkit_file\tchr\ttarget_length\tposition\tcontext\tmean_methylation\tn_loci\tn_measurements",
-                    "emseq-test1\tchr1\t182\t178\tCG\t0\t1\t1",
-                    "emseq-test1\tchr1\t204\t189\tCG\t0\t1\t1"
+                    "emseq-test1.ds.1\tchr1\t208\t110\tCG\t100\t1\t1",
+                    "emseq-test1.ds.1\tchr1\t204\t189\tCG\t33.3333\t1\t1",
+                    "emseq-test1.ds.1\tchr1\t151\t142\tCG\t100\t1\t1",
+                    "emseq-test1.ds.1\tchr1\t182\t178\tCG\t33.3333\t1\t1"
                 ]
             ],
             [
@@ -270,17 +260,13 @@
             [
                 "region_summary",
                 [
-                    "library\ttarget_locus\ttarget_name\tcontext\tmean_methylation",
-                    "emseq-test2\tchr1:140839-141043\tStemLoop_140889_140992_2mm_22C_0CG\tCG\t0",
-                    "emseq-test2\tchr1:140850-141032\tStemLoop_140900_140981_0mm_29C_1CG\tCG\t0"
+                    "library\ttarget_locus\ttarget_name\tcontext\tmean_methylation"
                 ]
             ],
             [
                 "methylkit_intersections",
                 [
-                    "methylkit_file\tchr\tstart\tend\tcontext\tmethylation\ttarget_locus\ttarget_name",
-                    "emseq-test2\tchr1\t141027\t141028\tCG\t0\tchr1:140840-141043\tStemLoop_140889_140992_2mm_22C_0CG",
-                    "emseq-test2\tchr1\t141027\t141028\tCG\t0\tchr1:140851-141032\tStemLoop_140900_140981_0mm_29C_1CG"
+                    "methylkit_file\tchr\tstart\tend\tcontext\tmethylation\ttarget_locus\ttarget_name"
                 ]
             ],
             [
@@ -294,36 +280,36 @@
             [
                 "ngs_agg",
                 [
-                    "--bam emseq-test1.md.bam ",
-                    "--bai emseq-test1.md.bai ",
-                    "--metadata_bam_file emseq-test1.bam ",
-                    "--fastp emseq-test1.fastp.json ",
-                    "--aln emseq-test1.alignment_summary_metrics.txt ",
-                    "--gc emseq-test1.gc_metrics ",
-                    "--dup emseq-test1.markdups_log ",
-                    "--idx_stats emseq-test1.idxstat ",
-                    "--flagstat emseq-test1.flagstat ",
-                    "--fastqc emseq-test1.md_fastqc.zip ",
-                    "--nonconverted_read_counts emseq-test1.nonconverted_counts.for_agg.tsv ",
-                    "--tasmanian emseq-test1.tasmanian.csv ",
-                    "--combined_mbias_records emseq-test1.combined_mbias.tsv ",
-                    "--fgbio_switchbacks emseq-test1.switchbacks.summary.txt",
+                    "--bam emseq-test1.ds.1.md.bam ",
+                    "--bai emseq-test1.ds.1.md.bai ",
+                    "--metadata_bam_file emseq-test1.ds.1.bam ",
+                    "--fastp emseq-test1.ds.1.fastp.json ",
+                    "--aln emseq-test1.ds.1.alignment_summary_metrics.txt ",
+                    "--gc emseq-test1.ds.1.gc_metrics ",
+                    "--dup emseq-test1.ds.1.markdups_log ",
+                    "--idx_stats emseq-test1.ds.1.idxstat ",
+                    "--flagstat emseq-test1.ds.1.flagstat ",
+                    "--fastqc emseq-test1.ds.1.md_fastqc.zip ",
+                    "--nonconverted_read_counts emseq-test1.ds.1.nonconverted_counts.for_agg.tsv ",
+                    "--tasmanian emseq-test1.ds.1.tasmanian.csv ",
+                    "--combined_mbias_records emseq-test1.ds.1.combined_mbias.tsv ",
+                    "--fgbio_switchbacks emseq-test1.ds.1.switchbacks.summary.txt",
                     "--workflow EM-seq"
                 ]
             ]
         ],
+        "timestamp": "2026-09-06T00:59:18.912472896",
         "meta": {
-            "nf-test": "0.9.5",
-            "nextflow": "24.10.5"
-        },
-        "timestamp": "2026-08-20T15:14:15.067200906"
+            "nf-test": "0.9.4",
+            "nextflow": "25.10.4"
+        }
     },
     "EM-seq workflow uBam": {
         "content": [
             {
-                "tasksCount": 49,
+                "tasksCount": 45,
                 "tasksFailed": 0,
-                "tasksSucceeded": 49
+                "tasksSucceeded": 45
             },
             [
                 "bwameth_file_1",
@@ -357,7 +343,7 @@
                 "fastp",
                 [
                     "\t\"summary\": {",
-                    "\t\t\"fastp_version\": \"1.3.6\",",
+                    "\t\t\"fastp_version\": \"0.1.0\",",
                     "\t\t\"sequencing\": \"paired end (76 cycles + 76 cycles)\",",
                     "\t\t\"before_filtering\": {",
                     "\t\t\t\"total_reads\": 9534,",
@@ -368,18 +354,18 @@
                     "\t\t\t\"q30_rate\": 0.913898,",
                     "\t\t\t\"read1_mean_length\": 76,",
                     "\t\t\t\"read2_mean_length\": 76,",
-                    "\t\t\t\"gc_content\": 0.218667",
+                    "\t\t\t\"gc_content\": 0.218668",
                     "\t\t},",
                     "\t\t\"after_filtering\": {",
                     "\t\t\t\"total_reads\": 9526,",
-                    "\t\t\t\"total_bases\": 719151,",
-                    "\t\t\t\"q20_bases\": 676526,",
-                    "\t\t\t\"q30_bases\": 657902,",
-                    "\t\t\t\"q20_rate\": 0.940729,",
-                    "\t\t\t\"q30_rate\": 0.914832,",
+                    "\t\t\t\"total_bases\": 719317,",
+                    "\t\t\t\"q20_bases\": 676675,",
+                    "\t\t\t\"q30_bases\": 658048,",
+                    "\t\t\t\"q20_rate\": 0.940719,",
+                    "\t\t\t\"q30_rate\": 0.914823,",
                     "\t\t\t\"read1_mean_length\": 75,",
                     "\t\t\t\"read2_mean_length\": 75,",
-                    "\t\t\t\"gc_content\": 0.216787",
+                    "\t\t\t\"gc_content\": 0.216795",
                     "\t\t}",
                     "\t},",
                     "\t\"filtering_result\": {",
@@ -429,20 +415,20 @@
             [
                 "flagstats",
                 [
-                    "1215 + 584 in total (QC-passed reads + QC-failed reads)",
-                    "1214 + 584 primary",
+                    "1799 + 0 in total (QC-passed reads + QC-failed reads)",
+                    "1798 + 0 primary",
                     "1 + 0 secondary",
                     "0 + 0 supplementary",
-                    "22 + 2 duplicates",
-                    "22 + 2 primary duplicates",
-                    "956 + 579 mapped (78.68% : 99.14%)",
-                    "955 + 579 primary mapped (78.67% : 99.14%)",
-                    "1214 + 584 paired in sequencing",
-                    "607 + 292 read1",
-                    "607 + 292 read2",
-                    "674 + 0 properly paired (55.52% : 0.00%)",
-                    "696 + 574 with itself and mate mapped",
-                    "259 + 5 singletons (21.33% : 0.86%)",
+                    "24 + 0 duplicates",
+                    "24 + 0 primary duplicates",
+                    "1535 + 0 mapped (85.33% : N/A)",
+                    "1534 + 0 primary mapped (85.32% : N/A)",
+                    "1798 + 0 paired in sequencing",
+                    "899 + 0 read1",
+                    "899 + 0 read2",
+                    "674 + 0 properly paired (37.49% : N/A)",
+                    "1270 + 0 with itself and mate mapped",
+                    "264 + 0 singletons (14.68% : N/A)",
                     "0 + 0 with mate mapped to a different chr",
                     "0 + 0 with mate mapped to a different chr (mapQ>=5)"
                 ]
@@ -558,9 +544,9 @@
                 "alignment_metrics",
                 [
                     "CATEGORY\tTOTAL_READS\tPF_READS\tPCT_PF_READS\tPF_NOISE_READS\tPF_READS_ALIGNED\tPCT_PF_READS_ALIGNED\tPF_ALIGNED_BASES\tPF_HQ_ALIGNED_READS\tPF_HQ_ALIGNED_BASES\tPF_HQ_ALIGNED_Q20_BASES\tPF_HQ_MEDIAN_MISMATCHES\tPF_MISMATCH_RATE\tPF_HQ_ERROR_RATE\tPF_INDEL_RATE\tMEAN_READ_LENGTH\tSD_READ_LENGTH\tMEDIAN_READ_LENGTH\tMAD_READ_LENGTH\tMIN_READ_LENGTH\tMAX_READ_LENGTH\tMEAN_ALIGNED_READ_LENGTH\tREADS_ALIGNED_IN_PAIRS\tPCT_READS_ALIGNED_IN_PAIRS\tPF_READS_IMPROPER_PAIRS\tPCT_PF_READS_IMPROPER_PAIRS\tBAD_CYCLES\tSTRAND_BALANCE\tPCT_CHIMERAS\tPCT_ADAPTER\tPCT_SOFTCLIP\tPCT_HARDCLIP\tAVG_POS_3PRIME_SOFTCLIP_LENGTH\tSAMPLE\tLIBRARY\tREAD_GROUP",
-                    "FIRST_OF_PAIR\t899\t607\t0.675195\t0\t493\t0.812191\t32309\t302\t19684\t18772\t3\t0.103312\t0.098954\t0.004364\t74.663921\t5.91042\t76\t0\t31\t76\t53.443163\t348\t0.705882\t156\t0.31643\t0\t0.89858\t0.010101\t0\t0.093312\t0\t15.835294\t\t\t",
-                    "SECOND_OF_PAIR\t899\t607\t0.675195\t0\t462\t0.76112\t30277\t299\t19999\t18469\t3\t0.102474\t0.09557\t0.004096\t74.710049\t5.864669\t76\t0\t31\t76\t50.116969\t348\t0.753247\t125\t0.270563\t0\t0.112554\t0.010453\t0\t0.086176\t0\t14.56701\t\t\t",
-                    "PAIR\t1798\t1214\t0.675195\t0\t955\t0.786656\t62586\t601\t39683\t37241\t3\t0.102909\t0.097258\t0.004234\t74.686985\t5.885207\t76\t0\t31\t76\t51.780066\t696\t0.728796\t281\t0.294241\t0\t0.518325\t0.010274\t0\t0.089743\t0\t15.159341\t\t\t"
+                    "FIRST_OF_PAIR\t899\t899\t1\t0\t781\t0.868743\t42522\t344\t22297\t21272\t0\t0.109413\t0.102292\t0.005362\t74.83426\t5.220772\t76\t0\t31\t76\t47.575083\t635\t0.81306\t444\t0.568502\t0\t0.806658\t0.010101\t0\t0.231137\t0\t23.036866\t\t\t",
+                    "SECOND_OF_PAIR\t899\t899\t1\t3\t753\t0.837597\t40929\t343\t22862\t21179\t0\t0.110193\t0.097406\t0.004984\t74.865406\t5.184858\t76\t0\t31\t76\t45.799778\t635\t0.843293\t416\t0.552457\t0\t0.203187\t0.010453\t0\t0.223375\t0\t20.209016\t\t\t",
+                    "PAIR\t1798\t1798\t1\t3\t1534\t0.85317\t83451\t687\t45159\t42451\t0\t0.109795\t0.09983\t0.005177\t74.849833\t5.201422\t76\t0\t31\t76\t46.68743\t1270\t0.827901\t860\t0.560626\t0\t0.51043\t0.010274\t0\t0.227255\t0\t21.54013\t\t\t"
                 ]
             ],
             [
@@ -674,10 +660,10 @@
                 ]
             ]
         ],
+        "timestamp": "2026-09-06T14:50:43.584038567",
         "meta": {
-            "nf-test": "0.9.5",
-            "nextflow": "24.10.5"
-        },
-        "timestamp": "2026-08-20T15:13:32.113269349"
+            "nf-test": "0.9.4",
+            "nextflow": "25.10.4"
+        }
     }
 }
\ No newline at end of file

From 0a2281f3fc967a5cd7687a162ec04396d095d83e Mon Sep 17 00:00:00 2001
From: Brad Langhorst <langhorst@neb.com>
Date: Mon, 7 Sep 2026 22:44:14 -0400
Subject: [PATCH 2/4] adds test fixtures until bamadap and picodup are
 published

---
 conf/test.config                              |    4 +-
 modules/merge_and_picodup.nf                  |   11 +
 modules/trim_and_align.nf                     |   15 +
 tests/fastq_to_ubam.nf.test.snap              |   30 +-
 tests/fixtures/reference_files/reference.dict |    2 +
 .../emseq-test1.ds.1.markdups_log             |   10 +
 .../merge_and_picodup/emseq-test1.ds.1.md.bai |  Bin 0 -> 472 bytes
 .../merge_and_picodup/emseq-test1.ds.1.md.bam |  Bin 0 -> 105276 bytes
 .../emseq-test1.markdups_log                  |   13 +
 .../merge_and_picodup/emseq-test1.md.bai      |  Bin 0 -> 472 bytes
 .../merge_and_picodup/emseq-test1.md.bam      |  Bin 0 -> 171814 bytes
 .../emseq-test2.ds.1.markdups_log             |   10 +
 .../merge_and_picodup/emseq-test2.ds.1.md.bai |  Bin 0 -> 560 bytes
 .../merge_and_picodup/emseq-test2.ds.1.md.bam |  Bin 0 -> 5831 bytes
 .../emseq-test2.markdups_log                  |   13 +
 .../merge_and_picodup/emseq-test2.md.bai      |  Bin 0 -> 472 bytes
 .../merge_and_picodup/emseq-test2.md.bam      |  Bin 0 -> 171882 bytes
 .../emseq-test1.ds.1_0_257200.aln.bam         |  Bin 0 -> 103945 bytes
 .../emseq-test1.ds.1_0_257200.fastp.json      | 5510 +++++++++++
 ...est1.ds.1_0_257200.nonconverted_counts.tsv |    1 +
 .../emseq-test1_0_250000.aln.bam              |  Bin 0 -> 102989 bytes
 .../emseq-test1_0_250000.fastp.json           | 8219 +++++++++++++++++
 ...seq-test1_0_250000.nonconverted_counts.tsv |    1 +
 .../emseq-test1_250000_421605.aln.bam         |  Bin 0 -> 66848 bytes
 .../emseq-test1_250000_421605.fastp.json      | 8205 ++++++++++++++++
 ...est1_250000_421605.nonconverted_counts.tsv |    1 +
 .../emseq-test2.ds.1_0_16821.aln.bam          |  Bin 0 -> 5593 bytes
 .../emseq-test2.ds.1_0_16821.fastp.json       | 4387 +++++++++
 ...test2.ds.1_0_16821.nonconverted_counts.tsv |    1 +
 .../emseq-test2_0_250000.aln.bam              |  Bin 0 -> 103099 bytes
 .../emseq-test2_0_250000.fastp.json           | 8219 +++++++++++++++++
 ...seq-test2_0_250000.nonconverted_counts.tsv |    1 +
 .../emseq-test2_250000_426231.aln.bam         |  Bin 0 -> 66869 bytes
 .../emseq-test2_250000_426231.fastp.json      | 8205 ++++++++++++++++
 ...est2_250000_426231.nonconverted_counts.tsv |    1 +
 35 files changed, 42843 insertions(+), 16 deletions(-)
 create mode 100644 tests/fixtures/reference_files/reference.dict
 create mode 100644 tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.ds.1.markdups_log
 create mode 100644 tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.ds.1.md.bai
 create mode 100644 tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.ds.1.md.bam
 create mode 100644 tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.markdups_log
 create mode 100644 tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.md.bai
 create mode 100644 tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.md.bam
 create mode 100644 tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.ds.1.markdups_log
 create mode 100644 tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.ds.1.md.bai
 create mode 100644 tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.ds.1.md.bam
 create mode 100644 tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.markdups_log
 create mode 100644 tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.md.bai
 create mode 100644 tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.md.bam
 create mode 100644 tests/fixtures/stub_outputs/trim_and_align/emseq-test1.ds.1_0_257200.aln.bam
 create mode 100644 tests/fixtures/stub_outputs/trim_and_align/emseq-test1.ds.1_0_257200.fastp.json
 create mode 100644 tests/fixtures/stub_outputs/trim_and_align/emseq-test1.ds.1_0_257200.nonconverted_counts.tsv
 create mode 100644 tests/fixtures/stub_outputs/trim_and_align/emseq-test1_0_250000.aln.bam
 create mode 100644 tests/fixtures/stub_outputs/trim_and_align/emseq-test1_0_250000.fastp.json
 create mode 100644 tests/fixtures/stub_outputs/trim_and_align/emseq-test1_0_250000.nonconverted_counts.tsv
 create mode 100644 tests/fixtures/stub_outputs/trim_and_align/emseq-test1_250000_421605.aln.bam
 create mode 100644 tests/fixtures/stub_outputs/trim_and_align/emseq-test1_250000_421605.fastp.json
 create mode 100644 tests/fixtures/stub_outputs/trim_and_align/emseq-test1_250000_421605.nonconverted_counts.tsv
 create mode 100644 tests/fixtures/stub_outputs/trim_and_align/emseq-test2.ds.1_0_16821.aln.bam
 create mode 100644 tests/fixtures/stub_outputs/trim_and_align/emseq-test2.ds.1_0_16821.fastp.json
 create mode 100644 tests/fixtures/stub_outputs/trim_and_align/emseq-test2.ds.1_0_16821.nonconverted_counts.tsv
 create mode 100644 tests/fixtures/stub_outputs/trim_and_align/emseq-test2_0_250000.aln.bam
 create mode 100644 tests/fixtures/stub_outputs/trim_and_align/emseq-test2_0_250000.fastp.json
 create mode 100644 tests/fixtures/stub_outputs/trim_and_align/emseq-test2_0_250000.nonconverted_counts.tsv
 create mode 100644 tests/fixtures/stub_outputs/trim_and_align/emseq-test2_250000_426231.aln.bam
 create mode 100644 tests/fixtures/stub_outputs/trim_and_align/emseq-test2_250000_426231.fastp.json
 create mode 100644 tests/fixtures/stub_outputs/trim_and_align/emseq-test2_250000_426231.nonconverted_counts.tsv

diff --git a/conf/test.config b/conf/test.config
index f814f87..724f57a 100644
--- a/conf/test.config
+++ b/conf/test.config
@@ -25,7 +25,9 @@ params {
 
     outputDir = "test_output"
 
-    // Not yet on bioconda; point the test profile at the local EL8 builds.
+    // Not yet on bioconda. nf-test's pipeline tests run trimAndAlign/mergeAndPicodup under
+    // -stub-run (see their stub: blocks), so CI never actually invokes these paths -- they
+    // only matter for a manual, non-stub `nextflow run main.nf -profile test`.
     bamadap_bin = '/appdev/langhorst/src/bamadap/target-el8/release/bamadap'
     picodup_bin = '/appdev/langhorst/src/picodup/target-el8/release/picodup'
     }
diff --git a/modules/merge_and_picodup.nf b/modules/merge_and_picodup.nf
index 92ba103..0890717 100644
--- a/modules/merge_and_picodup.nf
+++ b/modules/merge_and_picodup.nf
@@ -39,4 +39,15 @@ process mergeAndPicodup {
 
     samtools index -@ ${task.cpus} ${library}.md.bam ${library}.md.bai
     """
+
+    stub:
+    // See the matching note in trim_and_align.nf: replays a real picodup run's output for
+    // this library, captured once under tests/fixtures/stub_outputs/, since bamadap/picodup
+    // aren't on bioconda yet and CI has no real binary to run.
+    def fixture_dir = "${workflow.projectDir}/tests/fixtures/stub_outputs/merge_and_picodup"
+    """
+    cp ${fixture_dir}/${library}.md.bam .
+    cp ${fixture_dir}/${library}.md.bai .
+    cp ${fixture_dir}/${library}.markdups_log .
+    """
 }
diff --git a/modules/trim_and_align.nf b/modules/trim_and_align.nf
index 0df3b20..d727bed 100644
--- a/modules/trim_and_align.nf
+++ b/modules/trim_and_align.nf
@@ -56,4 +56,19 @@ process trimAndAlign {
 
     rm -f ${slice_path}
     """
+
+    stub:
+    chunk = "${library}_${start_offset}_${end_offset}"
+    // bamadap/picodup aren't on bioconda yet, so CI can't install real binaries; -stub-run
+    // instead replays real output this exact chunk produced on a genuine (local-machine)
+    // run of the real pipeline against these same fixtures, captured once under
+    // tests/fixtures/stub_outputs/. Regenerate those fixtures if trim_and_align.nf's
+    // logic, its conda pins, or the test fixtures themselves change.
+    def fixture_dir = "${workflow.projectDir}/tests/fixtures/stub_outputs/trim_and_align"
+    """
+    cp ${fixture_dir}/${chunk}.aln.bam .
+    cp ${fixture_dir}/${chunk}.fastp.json .
+    cp ${fixture_dir}/${chunk}.nonconverted_counts.tsv .
+    touch ${library}.log.bwamem
+    """
 }
diff --git a/tests/fastq_to_ubam.nf.test.snap b/tests/fastq_to_ubam.nf.test.snap
index ba86327..545084b 100644
--- a/tests/fastq_to_ubam.nf.test.snap
+++ b/tests/fastq_to_ubam.nf.test.snap
@@ -2,9 +2,9 @@
     "fastq to uBam workflow - single-end": {
         "content": [
             {
-                "tasksCount": 2,
+                "tasksCount": 4,
                 "tasksFailed": 0,
-                "tasksSucceeded": 2
+                "tasksSucceeded": 4
             },
             [
                 "ubam",
@@ -19,18 +19,18 @@
                 }
             ]
         ],
-        "timestamp": "2026-07-29T14:20:52.691934926",
+        "timestamp": "2026-09-07T22:24:27.574255349",
         "meta": {
-            "nf-test": "0.9.5",
-            "nextflow": "26.04.0"
+            "nf-test": "0.9.4",
+            "nextflow": "25.10.4"
         }
     },
     "fastq to uBam workflow - non-standard SRA headers (issue #15)": {
         "content": [
             {
-                "tasksCount": 1,
+                "tasksCount": 2,
                 "tasksFailed": 0,
-                "tasksSucceeded": 1
+                "tasksSucceeded": 2
             },
             [
                 "ubam",
@@ -45,18 +45,18 @@
                 }
             ]
         ],
-        "timestamp": "2026-07-29T14:20:59.43877386",
+        "timestamp": "2026-09-07T22:24:36.675298676",
         "meta": {
-            "nf-test": "0.9.5",
-            "nextflow": "26.04.0"
+            "nf-test": "0.9.4",
+            "nextflow": "25.10.4"
         }
     },
     "fastq to uBam workflow - paired-end": {
         "content": [
             {
-                "tasksCount": 2,
+                "tasksCount": 4,
                 "tasksFailed": 0,
-                "tasksSucceeded": 2
+                "tasksSucceeded": 4
             },
             [
                 "ubam",
@@ -71,10 +71,10 @@
                 }
             ]
         ],
-        "timestamp": "2026-07-29T14:20:45.730120318",
+        "timestamp": "2026-09-07T22:24:18.57790468",
         "meta": {
-            "nf-test": "0.9.5",
-            "nextflow": "26.04.0"
+            "nf-test": "0.9.4",
+            "nextflow": "25.10.4"
         }
     }
 }
\ No newline at end of file
diff --git a/tests/fixtures/reference_files/reference.dict b/tests/fixtures/reference_files/reference.dict
new file mode 100644
index 0000000..918b1b0
--- /dev/null
+++ b/tests/fixtures/reference_files/reference.dict
@@ -0,0 +1,2 @@
+@HD	VN:1.0	SO:unsorted
+@SQ	SN:chr1	LN:284940	M5:e2d98a7cc072e12f74f7ef08529c1cba	UR:file:///appdev/langhorst/src/EM-seq/tests/fixtures/reference_files/reference.fa
diff --git a/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.ds.1.markdups_log b/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.ds.1.markdups_log
new file mode 100644
index 0000000..10ff3b3
--- /dev/null
+++ b/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.ds.1.markdups_log
@@ -0,0 +1,10 @@
+## htsjdk.samtools.metrics.StringHeader
+# /appdev/langhorst/src/picodup/target-el8/release/picodup --input /dev/stdin --output emseq-test1.ds.1.md.bam --metrics emseq-test1.ds.1.markdups_log --threads 14 --optical-model ellipse --optical-distance 2500 --tagging-policy All
+## htsjdk.samtools.metrics.StringHeader
+# Started on: Mon Sep 07 22:25:49 EDT 2026
+
+## METRICS CLASS	picard.sam.DuplicationMetrics
+LIBRARY	UNPAIRED_READS_EXAMINED	READ_PAIRS_EXAMINED	SECONDARY_OR_SUPPLEMENTARY_RDS	UNMAPPED_READS	UNPAIRED_READ_DUPLICATES	READ_PAIR_DUPLICATES	READ_PAIR_OPTICAL_DUPLICATES	PERCENT_DUPLICATION	ESTIMATED_LIBRARY_SIZE
+emseq-test1.ds.1	745	0	18	0	14	0	0	0.018792	
+
+
diff --git a/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.ds.1.md.bai b/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.ds.1.md.bai
new file mode 100644
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diff --git a/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.ds.1.md.bam b/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.ds.1.md.bam
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diff --git a/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.markdups_log b/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.markdups_log
new file mode 100644
index 0000000..507e936
--- /dev/null
+++ b/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.markdups_log
@@ -0,0 +1,13 @@
+## htsjdk.samtools.metrics.StringHeader
+# /appdev/langhorst/src/picodup/target-el8/release/picodup --input /dev/stdin --output emseq-test1.md.bam --metrics emseq-test1.markdups_log --threads 14 --optical-model ellipse --optical-distance 100 --tagging-policy All
+## htsjdk.samtools.metrics.StringHeader
+# Started on: Mon Sep 07 22:25:29 EDT 2026
+
+## METRICS CLASS	picard.sam.DuplicationMetrics
+LIBRARY	UNPAIRED_READS_EXAMINED	READ_PAIRS_EXAMINED	SECONDARY_OR_SUPPLEMENTARY_RDS	UNMAPPED_READS	UNPAIRED_READ_DUPLICATES	READ_PAIR_DUPLICATES	READ_PAIR_OPTICAL_DUPLICATES	PERCENT_DUPLICATION	ESTIMATED_LIBRARY_SIZE
+Oct25_PV_10ngR3_F6	264	635	1	264	24	0	0	0.015645	
+
+## HISTOGRAM	java.lang.Double
+BIN	CoverageMult	all_sets	optical_sets	non_optical_sets
+1.0	0.000000	623	0	623
+
diff --git a/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.md.bai b/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.md.bai
new file mode 100644
index 0000000000000000000000000000000000000000..cc29ca92e85b2de9a16bbddbe7a99d1212484db7
GIT binary patch
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literal 0
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diff --git a/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.md.bam b/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test1.md.bam
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diff --git a/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.ds.1.markdups_log b/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.ds.1.markdups_log
new file mode 100644
index 0000000..8b1b551
--- /dev/null
+++ b/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.ds.1.markdups_log
@@ -0,0 +1,10 @@
+## htsjdk.samtools.metrics.StringHeader
+# /appdev/langhorst/src/picodup/target-el8/release/picodup --input /dev/stdin --output emseq-test2.ds.1.md.bam --metrics emseq-test2.ds.1.markdups_log --threads 14 --optical-model ellipse --optical-distance 2500 --tagging-policy All
+## htsjdk.samtools.metrics.StringHeader
+# Started on: Mon Sep 07 22:25:49 EDT 2026
+
+## METRICS CLASS	picard.sam.DuplicationMetrics
+LIBRARY	UNPAIRED_READS_EXAMINED	READ_PAIRS_EXAMINED	SECONDARY_OR_SUPPLEMENTARY_RDS	UNMAPPED_READS	UNPAIRED_READ_DUPLICATES	READ_PAIR_DUPLICATES	READ_PAIR_OPTICAL_DUPLICATES	PERCENT_DUPLICATION	ESTIMATED_LIBRARY_SIZE
+emseq-test2.ds.1	48	0	0	0	0	0	0	0.000000	
+
+
diff --git a/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.ds.1.md.bai b/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.ds.1.md.bai
new file mode 100644
index 0000000000000000000000000000000000000000..f094e5f7c1e47db7cd6e630a856be4825001577e
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diff --git a/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.ds.1.md.bam b/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.ds.1.md.bam
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diff --git a/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.markdups_log b/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.markdups_log
new file mode 100644
index 0000000..e6b775e
--- /dev/null
+++ b/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.markdups_log
@@ -0,0 +1,13 @@
+## htsjdk.samtools.metrics.StringHeader
+# /appdev/langhorst/src/picodup/target-el8/release/picodup --input /dev/stdin --output emseq-test2.md.bam --metrics emseq-test2.markdups_log --threads 14 --optical-model ellipse --optical-distance 100 --tagging-policy All
+## htsjdk.samtools.metrics.StringHeader
+# Started on: Mon Sep 07 22:25:29 EDT 2026
+
+## METRICS CLASS	picard.sam.DuplicationMetrics
+LIBRARY	UNPAIRED_READS_EXAMINED	READ_PAIRS_EXAMINED	SECONDARY_OR_SUPPLEMENTARY_RDS	UNMAPPED_READS	UNPAIRED_READ_DUPLICATES	READ_PAIR_DUPLICATES	READ_PAIR_OPTICAL_DUPLICATES	PERCENT_DUPLICATION	ESTIMATED_LIBRARY_SIZE
+Oct25_PV_10ngR3_F6	264	635	1	264	23	0	0	0.014993	
+
+## HISTOGRAM	java.lang.Double
+BIN	CoverageMult	all_sets	optical_sets	non_optical_sets
+1.0	0.000000	624	0	624
+
diff --git a/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.md.bai b/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.md.bai
new file mode 100644
index 0000000000000000000000000000000000000000..0fb78c3866c0ee83ab03c0aaef79f6f1ed7ada9d
GIT binary patch
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diff --git a/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.md.bam b/tests/fixtures/stub_outputs/merge_and_picodup/emseq-test2.md.bam
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diff --git a/tests/fixtures/stub_outputs/trim_and_align/emseq-test1.ds.1_0_257200.aln.bam b/tests/fixtures/stub_outputs/trim_and_align/emseq-test1.ds.1_0_257200.aln.bam
new file mode 100644
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diff --git a/tests/fixtures/stub_outputs/trim_and_align/emseq-test1.ds.1_0_257200.fastp.json b/tests/fixtures/stub_outputs/trim_and_align/emseq-test1.ds.1_0_257200.fastp.json
new file mode 100644
index 0000000..397f181
--- /dev/null
+++ b/tests/fixtures/stub_outputs/trim_and_align/emseq-test1.ds.1_0_257200.fastp.json
@@ -0,0 +1,5510 @@
+{
+  "summary": {
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+    "fastp_version": "0.1.0",
+    "sequencing": "single end (150 cycles)",
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+      "q30_bases": 356222,
+      "q20_rate": 0.9820906666666667,
+      "q30_rate": 0.9499253333333333,
+      "read1_mean_length": 150,
+      "gc_content": 0.21490933333333334
+    },
+    "after_filtering": {
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+      "total_bases": 371387,
+      "q20_bases": 364731,
+      "q30_bases": 352753,
+      "q20_rate": 0.9820779941139566,
+      "q30_rate": 0.9498259228244392,
+      "read1_mean_length": 148,
+      "gc_content": 0.21198103326179968
+    }
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+  "filtering_result": {
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+    "low_quality_reads": 0,
+    "too_many_N_reads": 0,
+    "adapter_dimer_reads": 0,
+    "too_short_reads": 0,
+    "too_long_reads": 0,
+    "low_complexity_reads": 0
+  },
+  "duplication": {
+    "rate": 0.0
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+  "adapter_cutting": {
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+    "adapter_trimmed_bases": 3613,
+    "read1_adapter_sequence": "AGATCGGAAGAGC",
+    "read2_adapter_sequence": "AGATCGGAAGAGC",
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+      "GGGGT": 0,
+      "GGGGC": 0,
+      "GGGGG": 0
+    },
+    "overrepresented_sequences": {}
+  },
+  "command": "/appdev/langhorst/src/bamadap/target-el8/release/bamadap --input /tmp/emseq-test1.ds.1_0_257200.slice.bam --format fastq --no-mate-suffix -l 2 --disable-quality-filtering --adapter-fasta illumina_adapters.fa --threads 4 --json emseq-test1.ds.1_0_257200.fastp.json"
+}
\ No newline at end of file
diff --git a/tests/fixtures/stub_outputs/trim_and_align/emseq-test1.ds.1_0_257200.nonconverted_counts.tsv b/tests/fixtures/stub_outputs/trim_and_align/emseq-test1.ds.1_0_257200.nonconverted_counts.tsv
new file mode 100644
index 0000000..4b1db9b
--- /dev/null
+++ b/tests/fixtures/stub_outputs/trim_and_align/emseq-test1.ds.1_0_257200.nonconverted_counts.tsv
@@ -0,0 +1 @@
+chr1	0
diff --git a/tests/fixtures/stub_outputs/trim_and_align/emseq-test1_0_250000.aln.bam b/tests/fixtures/stub_outputs/trim_and_align/emseq-test1_0_250000.aln.bam
new file mode 100644
index 0000000000000000000000000000000000000000..0f4388e2384e4b2bb18baf0c1b4067dc6660d402
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diff --git a/tests/fixtures/stub_outputs/trim_and_align/emseq-test1_0_250000.fastp.json b/tests/fixtures/stub_outputs/trim_and_align/emseq-test1_0_250000.fastp.json
new file mode 100644
index 0000000..0c4f080
--- /dev/null
+++ b/tests/fixtures/stub_outputs/trim_and_align/emseq-test1_0_250000.fastp.json
@@ -0,0 +1,8219 @@
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+      "GGTGG": 0,
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+      "GGCTA": 0,
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+      "GGGGA": 0,
+      "GGGGT": 0,
+      "GGGGC": 0,
+      "GGGGG": 0
+    },
+    "overrepresented_sequences": {}
+  },
+  "command": "/appdev/langhorst/src/bamadap/target-el8/release/bamadap --input /tmp/emseq-test1_0_250000.slice.bam --format fastq --no-mate-suffix -l 2 --disable-quality-filtering --adapter-fasta illumina_adapters.fa --trim-poly-g --threads 4 --json emseq-test1_0_250000.fastp.json"
+}
\ No newline at end of file
diff --git a/tests/fixtures/stub_outputs/trim_and_align/emseq-test1_0_250000.nonconverted_counts.tsv b/tests/fixtures/stub_outputs/trim_and_align/emseq-test1_0_250000.nonconverted_counts.tsv
new file mode 100644
index 0000000..4b1db9b
--- /dev/null
+++ b/tests/fixtures/stub_outputs/trim_and_align/emseq-test1_0_250000.nonconverted_counts.tsv
@@ -0,0 +1 @@
+chr1	0
diff --git a/tests/fixtures/stub_outputs/trim_and_align/emseq-test1_250000_421605.aln.bam b/tests/fixtures/stub_outputs/trim_and_align/emseq-test1_250000_421605.aln.bam
new file mode 100644
index 0000000000000000000000000000000000000000..734fe9041c58bf849f87234ed53b239b42bd7de7
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diff --git a/tests/fixtures/stub_outputs/trim_and_align/emseq-test1_250000_421605.fastp.json b/tests/fixtures/stub_outputs/trim_and_align/emseq-test1_250000_421605.fastp.json
new file mode 100644
index 0000000..4fcaf3b
--- /dev/null
+++ b/tests/fixtures/stub_outputs/trim_and_align/emseq-test1_250000_421605.fastp.json
@@ -0,0 +1,8205 @@
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+      "q30_bases": 268156,
+      "q20_rate": 0.9409260371999016,
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+      "read1_mean_length": 76,
+      "read2_mean_length": 76,
+      "gc_content": 0.21639513287630077
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+      "total_bases": 290833,
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+      "read1_mean_length": 75,
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+      "gc_content": 0.21431543188015115
+    }
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+  "filtering_result": {
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+      "GCGGG": 0,
+      "GGAAA": 0,
+      "GGAAT": 0,
+      "GGAAC": 0,
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+      "GGATT": 0,
+      "GGATC": 0,
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+      "GGACT": 0,
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+      "GGACG": 0,
+      "GGAGA": 0,
+      "GGAGT": 0,
+      "GGAGC": 0,
+      "GGAGG": 0,
+      "GGTAA": 0,
+      "GGTAT": 0,
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+      "GGTTA": 0,
+      "GGTTT": 0,
+      "GGTTC": 0,
+      "GGTTG": 0,
+      "GGTCA": 0,
+      "GGTCT": 0,
+      "GGTCC": 0,
+      "GGTCG": 0,
+      "GGTGA": 0,
+      "GGTGT": 0,
+      "GGTGC": 0,
+      "GGTGG": 0,
+      "GGCAA": 0,
+      "GGCAT": 0,
+      "GGCAC": 0,
+      "GGCAG": 0,
+      "GGCTA": 0,
+      "GGCTT": 0,
+      "GGCTC": 0,
+      "GGCTG": 0,
+      "GGCCA": 0,
+      "GGCCT": 0,
+      "GGCCC": 0,
+      "GGCCG": 0,
+      "GGCGA": 0,
+      "GGCGT": 0,
+      "GGCGC": 0,
+      "GGCGG": 0,
+      "GGGAA": 0,
+      "GGGAT": 0,
+      "GGGAC": 0,
+      "GGGAG": 0,
+      "GGGTA": 0,
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+      "GGGTG": 0,
+      "GGGCA": 0,
+      "GGGCT": 0,
+      "GGGCC": 0,
+      "GGGCG": 0,
+      "GGGGA": 0,
+      "GGGGT": 0,
+      "GGGGC": 0,
+      "GGGGG": 0
+    },
+    "overrepresented_sequences": {}
+  },
+  "command": "/appdev/langhorst/src/bamadap/target-el8/release/bamadap --input /tmp/emseq-test1_250000_421605.slice.bam --format fastq --no-mate-suffix -l 2 --disable-quality-filtering --adapter-fasta illumina_adapters.fa --trim-poly-g --threads 4 --json emseq-test1_250000_421605.fastp.json"
+}
\ No newline at end of file
diff --git a/tests/fixtures/stub_outputs/trim_and_align/emseq-test1_250000_421605.nonconverted_counts.tsv b/tests/fixtures/stub_outputs/trim_and_align/emseq-test1_250000_421605.nonconverted_counts.tsv
new file mode 100644
index 0000000..4b1db9b
--- /dev/null
+++ b/tests/fixtures/stub_outputs/trim_and_align/emseq-test1_250000_421605.nonconverted_counts.tsv
@@ -0,0 +1 @@
+chr1	0
diff --git a/tests/fixtures/stub_outputs/trim_and_align/emseq-test2.ds.1_0_16821.aln.bam b/tests/fixtures/stub_outputs/trim_and_align/emseq-test2.ds.1_0_16821.aln.bam
new file mode 100644
index 0000000000000000000000000000000000000000..ac12614bf82f1be4a0474d43b5e7c9629be27beb
GIT binary patch
literal 5593
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diff --git a/tests/fixtures/stub_outputs/trim_and_align/emseq-test2.ds.1_0_16821.fastp.json b/tests/fixtures/stub_outputs/trim_and_align/emseq-test2.ds.1_0_16821.fastp.json
new file mode 100644
index 0000000..9719ab6
--- /dev/null
+++ b/tests/fixtures/stub_outputs/trim_and_align/emseq-test2.ds.1_0_16821.fastp.json
@@ -0,0 +1,4387 @@
+{
+  "summary": {
+    "bamadap_version": "0.1.0",
+    "fastp_version": "0.1.0",
+    "sequencing": "single end (99 cycles)",
+    "before_filtering": {
+      "total_reads": 369,
+      "total_bases": 36531,
+      "q20_bases": 36142,
+      "q30_bases": 35164,
+      "q20_rate": 0.9893515096767129,
+      "q30_rate": 0.962579726807369,
+      "read1_mean_length": 99,
+      "gc_content": 0.2288741069228874
+    },
+    "after_filtering": {
+      "total_reads": 369,
+      "total_bases": 35186,
+      "q20_bases": 34807,
+      "q30_bases": 33870,
+      "q20_rate": 0.9892286704939465,
+      "q30_rate": 0.9625987608708009,
+      "read1_mean_length": 95,
+      "gc_content": 0.21767180128460184
+    }
+  },
+  "filtering_result": {
+    "passed_filter_reads": 369,
+    "low_quality_reads": 0,
+    "too_many_N_reads": 0,
+    "adapter_dimer_reads": 0,
+    "too_short_reads": 0,
+    "too_long_reads": 0,
+    "low_complexity_reads": 0
+  },
+  "duplication": {
+    "rate": 0.0
+  },
+  "adapter_cutting": {
+    "adapter_trimmed_reads": 60,
+    "adapter_trimmed_bases": 1345,
+    "read1_adapter_sequence": "AGATCGGAAGAGC",
+    "read2_adapter_sequence": "AGATCGGAAGAGC",
+    "read1_adapter_counts": {
+      "AGATCG": 4,
+      "AGATCGG": 3,
+      "AGATCGGAAG": 3,
+      "AGATCGGAAGA": 3,
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+      "AGATCGGAAGAGCACA": 2,
+      "AGATCGGAAGAGCACAC": 3,
+      "AGATCGGAAGAGCACACGTCT": 4,
+      "AGATCGGAAGAGCACACGTCTG": 2,
+      "AGATCGGAAGAGCACACGTCTGA": 2,
+      "AGATCGGAAGAGCACACGTCTGAAC": 4,
+      "AGATCGGAAGAGCACACGTCTGAACTCCA": 3,
+      "AGATCGGAAGAGCACACGTCTGAACTCCAGTCACTCG": 3,
+      "AGATCGGAAGAGCACACGTCTGAACTCCAGTCACTCGG": 3,
+      "AGATCGGAAGAGCACACGTCTGAACTCCAGTCACTCGGAT": 2,
+      "others": 17
+    },
+    "read2_adapter_counts": {
+      "others": 0
+    },
+    "extra_adapter_sequences": [
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+      "AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT"
+    ]
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+  "polyx_trimming": {
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+    "polyx_trimmed_reads": {
+      "A": 0,
+      "T": 0,
+      "C": 0,
+      "G": 0
+    },
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+      "GGGCC": 0,
+      "GGGCG": 0,
+      "GGGGA": 0,
+      "GGGGT": 0,
+      "GGGGC": 0,
+      "GGGGG": 0
+    },
+    "overrepresented_sequences": {}
+  },
+  "command": "/appdev/langhorst/src/bamadap/target-el8/release/bamadap --input /tmp/emseq-test2.ds.1_0_16821.slice.bam --format fastq --no-mate-suffix -l 2 --disable-quality-filtering --adapter-fasta illumina_adapters.fa --trim-poly-g --threads 4 --json emseq-test2.ds.1_0_16821.fastp.json"
+}
\ No newline at end of file
diff --git a/tests/fixtures/stub_outputs/trim_and_align/emseq-test2.ds.1_0_16821.nonconverted_counts.tsv b/tests/fixtures/stub_outputs/trim_and_align/emseq-test2.ds.1_0_16821.nonconverted_counts.tsv
new file mode 100644
index 0000000..4b1db9b
--- /dev/null
+++ b/tests/fixtures/stub_outputs/trim_and_align/emseq-test2.ds.1_0_16821.nonconverted_counts.tsv
@@ -0,0 +1 @@
+chr1	0
diff --git a/tests/fixtures/stub_outputs/trim_and_align/emseq-test2_0_250000.aln.bam b/tests/fixtures/stub_outputs/trim_and_align/emseq-test2_0_250000.aln.bam
new file mode 100644
index 0000000000000000000000000000000000000000..7d2de7a0778a60294c846ae1a8318a60d97936bd
GIT binary patch
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diff --git a/tests/fixtures/stub_outputs/trim_and_align/emseq-test2_0_250000.fastp.json b/tests/fixtures/stub_outputs/trim_and_align/emseq-test2_0_250000.fastp.json
new file mode 100644
index 0000000..27b66cf
--- /dev/null
+++ b/tests/fixtures/stub_outputs/trim_and_align/emseq-test2_0_250000.fastp.json
@@ -0,0 +1,8219 @@
+{
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+    "fastp_version": "0.1.0",
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+      "q30_bases": 393968,
+      "q20_rate": 0.9390891401037805,
+      "q30_rate": 0.9126389918458118,
+      "read1_mean_length": 76,
+      "read2_mean_length": 76,
+      "gc_content": 0.22025574499629355
+    },
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+      "total_bases": 428484,
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+      "read1_mean_length": 75,
+      "read2_mean_length": 75,
+      "gc_content": 0.21852391221142448
+    }
+  },
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+      "GGTTC": 0,
+      "GGTTG": 0,
+      "GGTCA": 0,
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+      "GGCAG": 0,
+      "GGCTA": 0,
+      "GGCTT": 0,
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+      "GGCTG": 0,
+      "GGCCA": 0,
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+      "GGCGT": 0,
+      "GGCGC": 0,
+      "GGCGG": 0,
+      "GGGAA": 0,
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+      "GGGAG": 0,
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+      "GGGCA": 0,
+      "GGGCT": 0,
+      "GGGCC": 0,
+      "GGGCG": 0,
+      "GGGGA": 0,
+      "GGGGT": 0,
+      "GGGGC": 0,
+      "GGGGG": 0
+    },
+    "overrepresented_sequences": {}
+  },
+  "command": "/appdev/langhorst/src/bamadap/target-el8/release/bamadap --input /tmp/emseq-test2_0_250000.slice.bam --format fastq --no-mate-suffix -l 2 --disable-quality-filtering --adapter-fasta illumina_adapters.fa --trim-poly-g --threads 4 --json emseq-test2_0_250000.fastp.json"
+}
\ No newline at end of file
diff --git a/tests/fixtures/stub_outputs/trim_and_align/emseq-test2_0_250000.nonconverted_counts.tsv b/tests/fixtures/stub_outputs/trim_and_align/emseq-test2_0_250000.nonconverted_counts.tsv
new file mode 100644
index 0000000..4b1db9b
--- /dev/null
+++ b/tests/fixtures/stub_outputs/trim_and_align/emseq-test2_0_250000.nonconverted_counts.tsv
@@ -0,0 +1 @@
+chr1	0
diff --git a/tests/fixtures/stub_outputs/trim_and_align/emseq-test2_250000_426231.aln.bam b/tests/fixtures/stub_outputs/trim_and_align/emseq-test2_250000_426231.aln.bam
new file mode 100644
index 0000000000000000000000000000000000000000..7d733708e3b3d60fc7d84ad747927aa26b37d69c
GIT binary patch
literal 66869
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z*n3s3%&dqNwW~6UAQBP`=%42s0!UZ{1PBVMbA3yGLjx6jclyses?ts3RYPp-cQs@q
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diff --git a/tests/fixtures/stub_outputs/trim_and_align/emseq-test2_250000_426231.fastp.json b/tests/fixtures/stub_outputs/trim_and_align/emseq-test2_250000_426231.fastp.json
new file mode 100644
index 0000000..a0fe1cd
--- /dev/null
+++ b/tests/fixtures/stub_outputs/trim_and_align/emseq-test2_250000_426231.fastp.json
@@ -0,0 +1,8205 @@
+{
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+    "fastp_version": "0.1.0",
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+      "total_bases": 292752,
+      "q20_bases": 275514,
+      "q30_bases": 268079,
+      "q20_rate": 0.9411173962944746,
+      "q30_rate": 0.9157204733016342,
+      "read1_mean_length": 76,
+      "read2_mean_length": 76,
+      "gc_content": 0.21632986281904137
+    },
+    "after_filtering": {
+      "total_reads": 3846,
+      "total_bases": 290681,
+      "q20_bases": 273848,
+      "q30_bases": 266484,
+      "q20_rate": 0.9420911583488429,
+      "q30_rate": 0.9167575452127934,
+      "read1_mean_length": 75,
+      "read2_mean_length": 75,
+      "gc_content": 0.21424860930022946
+    }
+  },
+  "filtering_result": {
+    "passed_filter_reads": 3846,
+    "low_quality_reads": 0,
+    "too_many_N_reads": 0,
+    "adapter_dimer_reads": 0,
+    "too_short_reads": 6,
+    "too_long_reads": 0,
+    "low_complexity_reads": 0
+  },
+  "duplication": {
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+      "GCCGC": 0,
+      "GCCGG": 0,
+      "GCGAA": 0,
+      "GCGAT": 0,
+      "GCGAC": 0,
+      "GCGAG": 0,
+      "GCGTA": 0,
+      "GCGTT": 0,
+      "GCGTC": 0,
+      "GCGTG": 0,
+      "GCGCA": 0,
+      "GCGCT": 0,
+      "GCGCC": 0,
+      "GCGCG": 0,
+      "GCGGA": 0,
+      "GCGGT": 0,
+      "GCGGC": 0,
+      "GCGGG": 0,
+      "GGAAA": 0,
+      "GGAAT": 0,
+      "GGAAC": 0,
+      "GGAAG": 0,
+      "GGATA": 0,
+      "GGATT": 0,
+      "GGATC": 0,
+      "GGATG": 0,
+      "GGACA": 0,
+      "GGACT": 0,
+      "GGACC": 0,
+      "GGACG": 0,
+      "GGAGA": 0,
+      "GGAGT": 0,
+      "GGAGC": 0,
+      "GGAGG": 0,
+      "GGTAA": 0,
+      "GGTAT": 0,
+      "GGTAC": 0,
+      "GGTAG": 0,
+      "GGTTA": 0,
+      "GGTTT": 0,
+      "GGTTC": 0,
+      "GGTTG": 0,
+      "GGTCA": 0,
+      "GGTCT": 0,
+      "GGTCC": 0,
+      "GGTCG": 0,
+      "GGTGA": 0,
+      "GGTGT": 0,
+      "GGTGC": 0,
+      "GGTGG": 0,
+      "GGCAA": 0,
+      "GGCAT": 0,
+      "GGCAC": 0,
+      "GGCAG": 0,
+      "GGCTA": 0,
+      "GGCTT": 0,
+      "GGCTC": 0,
+      "GGCTG": 0,
+      "GGCCA": 0,
+      "GGCCT": 0,
+      "GGCCC": 0,
+      "GGCCG": 0,
+      "GGCGA": 0,
+      "GGCGT": 0,
+      "GGCGC": 0,
+      "GGCGG": 0,
+      "GGGAA": 0,
+      "GGGAT": 0,
+      "GGGAC": 0,
+      "GGGAG": 0,
+      "GGGTA": 0,
+      "GGGTT": 0,
+      "GGGTC": 0,
+      "GGGTG": 0,
+      "GGGCA": 0,
+      "GGGCT": 0,
+      "GGGCC": 0,
+      "GGGCG": 0,
+      "GGGGA": 0,
+      "GGGGT": 0,
+      "GGGGC": 0,
+      "GGGGG": 0
+    },
+    "overrepresented_sequences": {}
+  },
+  "command": "/appdev/langhorst/src/bamadap/target-el8/release/bamadap --input /tmp/emseq-test2_250000_426231.slice.bam --format fastq --no-mate-suffix -l 2 --disable-quality-filtering --adapter-fasta illumina_adapters.fa --trim-poly-g --threads 4 --json emseq-test2_250000_426231.fastp.json"
+}
\ No newline at end of file
diff --git a/tests/fixtures/stub_outputs/trim_and_align/emseq-test2_250000_426231.nonconverted_counts.tsv b/tests/fixtures/stub_outputs/trim_and_align/emseq-test2_250000_426231.nonconverted_counts.tsv
new file mode 100644
index 0000000..4b1db9b
--- /dev/null
+++ b/tests/fixtures/stub_outputs/trim_and_align/emseq-test2_250000_426231.nonconverted_counts.tsv
@@ -0,0 +1 @@
+chr1	0

From eb34b88da7298c4d052bf41b4a1d81a127bd1d85 Mon Sep 17 00:00:00 2001
From: Brad Langhorst <langhorst@neb.com>
Date: Mon, 7 Sep 2026 22:47:44 -0400
Subject: [PATCH 3/4] CI dependency version updates

---
 .github/workflows/test.yml | 16 ++++++++--------
 1 file changed, 8 insertions(+), 8 deletions(-)

diff --git a/.github/workflows/test.yml b/.github/workflows/test.yml
index c8ba2c0..565293d 100644
--- a/.github/workflows/test.yml
+++ b/.github/workflows/test.yml
@@ -18,21 +18,21 @@ jobs:
 
     steps:
       - name: Checkout
-        uses: actions/checkout@v4
+        uses: actions/checkout@v7
 
       - name: Setup Micromamba
-        uses: mamba-org/setup-micromamba@v1
+        uses: mamba-org/setup-micromamba@v3
         with:
           micromamba-version: '1.5.10-0' # any version from https://github.com/mamba-org/micromamba-releases
           post-cleanup: 'all'
 
       - name: Setup Nextflow (minimum supported version)
-        uses: nf-core/setup-nextflow@v2
+        uses: nf-core/setup-nextflow@v3
         with:
           version: '25.04.0'
 
       - name: Install nf-test
-        uses: nf-core/setup-nf-test@v1
+        uses: nf-core/setup-nf-test@v2
         with:
           version: 0.9.5
 
@@ -49,21 +49,21 @@ jobs:
 
     steps:
       - name: Checkout
-        uses: actions/checkout@v4
+        uses: actions/checkout@v7
 
       - name: Setup Micromamba
-        uses: mamba-org/setup-micromamba@v1
+        uses: mamba-org/setup-micromamba@v3
         with:
           micromamba-version: '1.5.10-0' # any version from https://github.com/mamba-org/micromamba-releases
           post-cleanup: 'all'
 
       - name: Setup Nextflow (latest)
-        uses: nf-core/setup-nextflow@v2
+        uses: nf-core/setup-nextflow@v3
         with:
           version: latest
 
       - name: Install nf-test
-        uses: nf-core/setup-nf-test@v1
+        uses: nf-core/setup-nf-test@v2
         with:
           version: 0.9.5
 

From a23e5128acea9252c4462edae4c23cac6756f149 Mon Sep 17 00:00:00 2001
From: Brad Langhorst <langhorst@neb.com>
Date: Mon, 7 Sep 2026 23:01:26 -0400
Subject: [PATCH 4/4] don't try to use more cpus than are available...

---
 conf/base.config | 4 +++-
 1 file changed, 3 insertions(+), 1 deletion(-)

diff --git a/conf/base.config b/conf/base.config
index d34447c..957899a 100644
--- a/conf/base.config
+++ b/conf/base.config
@@ -26,7 +26,9 @@ process {
     withLabel: dedup {
         // Deliberately not capped by params.max_cpus: this label exists so the dedup stage
         // can be tuned independently of the trim/align concurrency (see BENCHMARK_PLAN.md phase 6).
-        cpus   = 16
+        // Still capped to whatever the machine actually has, so it doesn't request more cpus
+        // than any executor can ever grant (e.g. GitHub Actions' 4-core runners).
+        cpus   = { Math.min(16, Runtime.runtime.availableProcessors()) }
     }
 
     withLabel: low_cpu {